UC-NRLF 


LABORATORY  OUTLINE 


FOR 


General    Bacteriology 


MAIN  LmRAFJY.AGRICULTU 


LABORATORY  OUTLINE 

-FOR- 

GENERAL  BACTERIOLOGY 


LABORATORY  OUTLINE 


GENERAL  BACTERIOLOGY 

BY 

CONNECTICUT  AGRICULTURAL 
COLLEGE 

BACTERIOLOGICAL  DEPARTMENT 


SEPTEMBER  1922 


BIOLOGY 

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LIBRARY-AGRICULTURE 


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PREFACE 


The  exercises  and  descriptions  in  this  laboratory  manual  are 
intended  primarily  for  students  in  General  Bacteriology.  The 
work  outlined  can  be  covered  in  an  eighteen  week  period  having 
two  two-hour  laboratory  periods  a  week,  preferably  at  intervals 
of  two  and  five  days.  The  course  offered  at  this  institution  includes 
two  one-hour  lectures  a  week.  The  lectures  and  laboratory  work 
are  planned  as  far  as  possible  to  parallel  and  supplement  each 
other. 

Students  in  this  course  do  not  prepare  their  culture  media  or 
sterilize  their  glassware  for  we  feel  that  the  beginner,  in  bacteriology 
can  use  the  limited  time  more  advantageously  in  other  directions. 

The  description  of  bacterial  species  includes  some  pathogens 
but  only  non-pathogenic  organisms  are  used  in  this  course. 

The  glossary  of  bacteriological  terms  appended  will  be  found 
helpful  to  the  student  beginning  the  study  of  bacteriology. 

Storrs,  Conn.,  The  Authors. 

September,  1922. 


497750 


CONTENTS 

PART  I. 

The  microscope 

Measurement  of  bacteria 

Sterilization 

Lockers  and  equipment 

Examination  of  unstained  bacteria 

Gelatin 

Agar 

Melting  and  solidifying  points  of  agar  and  gelatin 

Air  analysis 

Cautions  and  directions  for  identification  of  unknown  cultures 

Descriptive  chart 

Plate  1.     Types  of  growth  in  stab  and  streak  cultures 

Age  for  examination  of  various  cultures 

Stains 

Morphology 

Descriptions  of  culture  media 

Descriptions  of  various  species  of  bacteria 


PART  II. 

Water  testing 

Bacteria  in  milk.    Plating  method 

Direct  microscopic  examination  of  milk 

Effect  of  temperature  on  keeping  quality  of  milk 

Bacteria  from  various  sources 

Distilled  water  experiment 

Bacteria  in  meats 

Testing  for  presence  of  spores 

Conversion  factors 

Comparison  of  metric  and  English  systems 

Glossary  of  laboratory  bacteriological  terms 


Part  I. 


THE  MICROSCOPE 

The  microscope  is  a  delicately  adjusted  instrument  and  great 
care  should  be  exercised  in  using  it  to  keep  it  in  good  condition. 
The  following  rules  should  be  observed: 

1.  When  not  in  use  keep  in  the  case.     Dust  causes  unusual 
wear  if  allowed  to  work  into  the  mechanism. 

2.  Alcohol  should  never  be  used  on  the  lacquered  parts.     If 
oily  material  is  to  be  removed  use  xylol  with  gentle  rubbing. 

3.  Should  the  stage  or  objectives  become  gummed  with  im- 
mersion oil  or  balsam  it  can  be  removed  with  lens  paper  moistened 
with  xylol. 

4.  Use  the  plane  mirror  for  daylight  and  the  concave  for  arti- 
ficial light. 

5.  Regulate  the  amount  of  light  by  raising  or  lowering  the 
Abbe  condenser.     This  is  done  with  a  screw  under  left  of  stage. 
The  higher  the  condenser  the  greater  the  amount  of  light.     Dust 
in  the  eyepiece  makes  the  field  appear  covered  with  specks.     Wipe 
with  lens  paper. 

6.  Do  not  touch  the  surface  of  the  lens  with  the  fingers. 

7.  Objectives  of  one  microscope  should  not  be  changed  to 
another  even  of  the  same  make. 

8.  To  focus  with  the  two  highest  powered  objectives  place 
object  to  be  examined  in  center  of  stage.     With  eyes  at  the  side 
at  the  level  of  the  stage  lower  the  objective  writh  the  coarse  adjust- 
ment until  it  nearly  touches  the  cover  glass.     Adjust  mirror  and 
condenser  to  give  desired  amount  of  light.     Focus  slowly  upward 
with  the  fine  adjustment  until  the  object  becomes  clear.    With  the 
1/12  or  oil  immersion  objective  a  drop  of  cedar  oil  between  the 
lens  and  the  cover  glass  is  necessary  to  prevent  dispersion  of  light. 

9.  The  fine  adjustment  is  used  for  bringing  out  details  in 
very  small  objects  consequently  has  a  limited  range  and  is  me- 
chanically delicate.    When  the  fine  adjustment  screw  stops  do  not 
force  it. 

10.  Never  use  oil  with  any  but  the  oil  immersion  objectives. 
With  Leitz  microscopes  the  1/12  objectives    (oil  immersion)   are 
copper  colored  with  nickel  end.    With  Bausch  &  Lomb  microscopes 
the  oil  immersions  are  nickeled  on  the  lower  half. 

11.  For  measuring  bacteria  a  finely  ruled  scale  is  used.    This 
is  placed  ruled  side  down  in  top  part  of  eyepiece.    The  eyepiece  may 
be  swung  around  to  make  the  scale  lie  in  any  direction  desired. 
There  are  ten  microns  between  each  of  the  numbers  on  the  scale. 

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Laboratory  Outline  for  General  Bacteriology 


All  apparent  measurements  have  to  be  corrected  by  multiplying 
by  a  microscope  constant.    This  varies  for  the  different  microscopes. 

12.  Clean  the  oil  immersion  objective  with  lens  paper  imme- 
diately after  using. 

13.  With  the  Bausch  &  Lomb  microscope  using  the  10X  eye- 
piece and  the  1/6  objective  the  magnification  is  430  and  the  working 
distance  is  .3  mm.    With  the  1/12  objective  the  magnification  is  950 
and  the  working  distance  is  .15  mm. 


MEASUREMENT  OF  BACTERIA 

For  measuring  bacteria  use  the  highest  power  objective — the 
1/12  oil  immersion.  When  an  object  is  magnified  1000  times  light 
is  diminished  at  the  same  rate  and  the  Abbe  condenser  is  necessary 
in  order  to  increase  the  light  sufficiently  to  see  the  object. 

Raise  the  condenser  as  high  as  possible.  It  is  regulated  by 
a  screw  at  the  left  side  under  the  stage. 

Place  a  drop  of  cedar  oil  on  the  preparation  to  be  examined. 
The  oil  has  the  same  refractive  index  as  the  glass  and  prevents 
dispersion  of  the  rays  of  light. 

Lower  the  objective  into  the  oil  while  looking  at  it  from  the 
side. 

Adjust  the  mirror  to  give  the  desired  amount  of  light.  Usually 
the  full  amount  is  best  unless  the  light  is  exceptionally  bright.  Use 
the  plane  mirror  for  daylight  and  the  concave  for  artificial  light. 

With  the  eye  at  the  microscope  focus  up  slowly  with  the  fine 
adjustment  until  the  object  shows  clearly. 

In  the  eyepiece  is  a  ruled  scale  by  which  bacteria  are  measured. 
The  eyepiece  is  ruled  to  1/10  millimeter.  The  space  between  num- 
bers on  the  scale  is  one  millimeter.  A  micron  is  1/1000  of  a  milli- 
meter or  1/1,000,000  of  a  meter,  which  magnified  by  the  microscope 
covers  about  .7  of  the  space  between  two  lines  on  the  eyepiece 
micrometer.  The  magnification  of  different  microscopes  varies 
slightly  so  all  have  been  tested  with  a  stage  micrometer  and  the 
factor  or  microscope  constant  determined.  This  microscope  con- 
stant is  marked  on  the  stage  of  the  microscope.  All  apparent 
measurements  have  to  be  multiplied  by  this  factor  to  get  the  actual 
measurements. 

The  following  four  stained  preparations  are  furnished : 
No.     19  B.  mesentericus 
32  Micrococcus  agilis 
114  Bact.  aerogenes 
84  Sarcina  flava 

Note  the  shape  and  arrangement  of  the  bacteria  and  measure 
their  size.  Bacteria  of  the  same  kind  vary  somewhat  in  size  so 

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Laboratory  Outline  for  General  Bacteriology 


measurements  of  the  smallest  and  largest  are  required  in  addition 
to  the  size  of  the  majority.  Measure  at  least  ten  of  the  medium 
sized  ones  and  average  them  in  finding  the  size  of  the  majority. 
If  bacteria  are  spherical  only  one  dimension — diameter — is  re- 
quired ;  if  rod  shaped  two  dimensions — length  and  width  are  re- 
quired. Measure  length  and  width  of  the  same  organisms. 

To  be  one  micron  in  length  the  bacterium  has  to  extend 
from  one  line  to  the  corresponding  point  on  the  next  line,  that  is, 
across  one  space  and  one  line.  The  lines  have  width  and  on  most 
of  the  scales  the  width  of  the  line  is  about  .3  micron  and  the  width 
of  the  space  .7  micron.  Now  if  a  bacterium  is  just  covered  by 
a  line  we  know  the  measurement  is  .3.  If  it  projects  beyond  the 
line  it  is  possible  to  make  an  accurate  estimate  of  the  proportion 
of  the  .7  covered  which  added  to  the  .3  gives  the  size;  or  if  it  just 
fills  the  space  between  two  lines  it  is  .7  micron.  The  scale  may 
be  moved  in  any  direction  by  turning  the  eyepiece  around. 

Multiply  apparent  measurements  by  the  microscope  constant 
and  record  results  in  the  following  form : 

For  Rods 

Smallest  Largest  Average 

Length  x  width  Length  x  width  Length  x  width 

Arrangement 


For  Cocci 

Largest 
Diameter 

Average 
Diameter 

Smallest 
Diameter 

Arrangement 

When  through  with  the  microscope  carefully  wipe  cedar  oil 
from  lens  with  lens  paper  furnished.     Also  remove  oil  from  slides. 


Laboratory  note  books  about  6%  X  S1^  inches  are  preferred. 
These  are  to  be  left  on  the  table  at  your  place.  They  are  not 
to  be  taken  from  the  laboratory. 


STERILIZATION 

Sterilization  is  the  destruction  of  all  forms  of  life.  It  may 
be  accomplished  in  various  ways.  Only  the  common  methods  of 
sterilization  by  heat  will  be  considered  here. 

Sterilization  in  a  naked  flame.  The  simplest  means  of  sterilizing 
a  metal  instrument  is  to  heat  it  in  the  flame.  This  method  is  usually 

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Laboratory  Outline  for  General  Bacteriology 


recommended  for  sterilizing  platinum  needles,  spatulas,  forceps, 
etc.  When  sterilizing  a  needle  heat  the  entire  length  of  it  to  redness 
in  the  flame.  When  sterilizing  hold  needle  vertically  in  flame.  Pass 
the  lower  part  of  the  handle  three  or  four  times  through  the  flame. 

Before  sterilization  a  platinum  needle  should  be  dried  by  hold- 
ing it  near  the  flame.  This  prevents  sputtering  which  scatters 
organisms,  especially  when  materials  such  as  fat  and  protein  are 
thrust  into  the  flame.  This  drying  is  of  special  importance  when 
working  with  pathogenic  material. 

Cool  the  sterilized  needle  before  using. 

Do  not  let  the  sterilized  needle  touch  anything  except  the  cul- 
ture from  which  the  inoculation  is  being  made  and  the  medium  to 
be  inoculated. 

Sterilize  needle  after  using  before  laying  it  down. 

By  passing  instruments  like  forceps  and  spatulas  several  times 
through  a  hot  flame  they  may  be  sterilized. 

Sterilization  by  hot  air.  The  usual  method  of  sterilizing  all 
glassware,  instruments  with  metal  handles,  etc.,  is  by  exposure  to 
hot  air.  A  temperature  of  160°  C.  should  be  maintained  for  one 
hour.  Heating  and  cooling  is  done  slowly  to  avoid  breaking  the 
glassware. 

Sterilization  by  steam.  Under  ten  pounds  pressure  for  fifteen 
minutes  in  an  autoclav  all  life  is  destroyed  unless  the  material 
is  in  large  bulk.  For  flasks  and  larger  volumes  of  liquid  the  period 
of  heating  is  prolonged  to  twenty  minutes.  The  temperature  thus 
obtained  is  from  114°  to  115°  C. 

Steam  without  pressure  at  100°  C.  is  used.  Materials  are 
heated  for  twenty  to  thirty  minutes  on  three  successive  days.  On 
the  first  day  the  vegetative  cells  are  killed.  The  spores  remaining 
develop  in  time  to  be  killed  by  the  heating  on  the  second  day.  Heat- 
ing on  the  third  day  is  to  make  certain  that  all  spores  have  had 
time  to  change  to  the  vegetative  stage  and  be  killed. 


LOCKERS  AND  EQUIPMENT 

Lockers  fitted  out  with  apparatus  needed  in  the  work  of  the 
course  are  assigned  to  students.  A  deposit  of  25c  is  required  for 
the  key.  This  is  refunded  at  the  end  of  the  course  when  the  key 
is  returned.  The  following  materials  are  in  each  locker : 

1  counting  plate 

1  hand  lens 

1  platinum  needle 

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Laboratory  Outline  for  General  Bacteriology 


1  platinum  loop 

1  test  tube  rack 

2  wire  baskets 
1  slide  box 

1  ruler 

1  thermometer 

2  pipette  cases 
50  Petri  dishes 
25  Ice  pipettes 

6  Ice  pipettes  graduated  to  1/10  cc. 

2  2cc  pipettes 

The  above  list  together  with  the  prices  is  on  the  inside  of  the 
locker  door.  Check  up  carefully  and  if  anything  is  missing  report 
at  once.  Absolutely  no  allowance  is  made  for  any  shortage  unless 
reported  at  this  time.  Each  student  is  held  responsible  for  these 
materials  and  will  be  charged  at  the  end  of  the  semester  for  any- 
thing broken  or  missing.  Do  not  leave  materials  out  on  the  tables 
or  lying  around  the  laboratory.  They  are  returned  when  the  owner- 
ship is  known  but  in  most  cases  it  is  not  known  and  the  student 
loses  them. 

The  Petri  dishes  or  plates  in  the  lockers  are  sterile  so  do  not 
open  them.  When  counting  pipettes  count  from  the  end  without 
taking  them  from  the  case,  as  they  are  sterilized  ready  for  use. 

Bring  a  towel  or  cloth  for  drying  glassware. 

Students  are  required  to  wash  their  Petri  dishes  and  pipettes 
after  using  them.  Other  glassware  is  cared  for  by  the  depart- 
ment. Care  must  be  used  to  wash  the  glassware  perfectly  clean. 
If  any  particles  of  media  remain,  sterilizing  burns  them  on  and 
leaves  dark  brown  patches  which  can  only  be  removed  by  scraping 
or  by  a  strong  acid  cleaning  mixture.  Petri  dishes  are  to  be  wiped 
dry.  Mark  each  one  with  your  locker  number.  All  glassware  is 
marked  with  red  glass  marking  pencils.  These  pencils  are  sold 
in  the  laboratory  for  lOc  each.  After  pipettes  are  cleaned  they 
are  returned  to  their  case  with  points  down.  Cases  are  also  marked 
with  the  locker  number.  Place  cases  of  pipettes  and  Petri  dishes 
which  you  wish  sterilized  on  the  long  table  in  north  end  of  labora- 
tory. In  the  book  there  write  your  name,  locker  number,  number 
of  dishes,  number  of  cases  of  pipettes  left  to  be  sterilized,  and  the 
date.  They  will  be  sterilized  and  returned  to  your  locker.  Plates 
and  pipettes  must  be  washed  promptly  when  you  are  through  using 
them. 

Forms  for  recording  notes  will  be  given  out  on  the  board.  Use 
the  forms  given.  Leave  one  page  in  the  note  book  for  each  experi- 
ment. 

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Laboratory  Outline  for  General  Bacteriology 


EXAMINATION  OF  UNSTAINED  BACTERIA 

The  purpose  is  to  study  bacteria  in  a  living  condition ;  to  dem- 
onstrate their  form,  arrangement,  and  motility. 

Bacteria  have  two  kinds  of  motion,  the  so-called  Brownian  or 
molecular  movement,  and  true  motility.  Brownian  movement  is 
shown  more  or  less  by  all  small  particles  of  insoluble  matter  (in- 
cluding living  non-motile  or  dead  bacteria)  in  suspension.  It  is 
characterized  by  a  vibratory  movement;  the  relative  positions  of 
the  particles  or  bacteria  are  not  changed.  This  type  of  movement 
may  be  distinguished  from  true  motility  which  is  characterized 
by  progressive  movement,  more  or  less  rapid,  of  an  organism  across 
the  field  of  a  microscope,  and  which  changes  its  position  in  the 
field  independently  of  and  in  a  direction  contrary  to  other  organisms 
present.  If  large  numbers  of  bacteria  are  moving  in  one  direction 
it  is  an  indication  that  they  are  being  carried  by  currents  in  the 
liquid. 

Method 

Place  a  drop  of  water  on  a  cover  glass  held  in  cover-glass 
forceps.  Sterilize  platinum  loop  by  heating  to  redness  in  gas  flame. 
When  cool  transfer  a  small  amount  of  the  culture  to  be  examined 
to  the  drop  of  water.  Use  only  enough  to  cloud  the  water  slightly. 
Take  the  culture  from  the  edge  of  the  growth  in  the  lower  part  of 
the  tube.  The  bacteria  are  on  the  surface  of  the  agar,  so  do  not 
allow  the  loop  to  cut  down  into  the  agar.  In  opening  the  tube 
remove  the  cotton  plug  and  hold  it  so  that  the  inner  part  will  not 
touch  anything.  Never  lay  it  on  the  table.  This  is  necessary  to 
prevent  the  culture  in  the  tube  from  becoming  contaminated  with 
other  kinds  of  bacteria  or  molds.  After  removing  loopful  of  cul- 
ture replace  cotton  plug.  Invert  the  preparation  onto  a  slide  and 
examine  with  the  1/6  objective.  No  oil  is  used  with  this  objective. 
With  the  coarse  adjustment  lower  the  objective  until  it  nearly 
touches  the  cover  glass,  being  careful  not  to  touch  it.  Then  with 
the  eye  at  the  ocular,  focus  up  with  the  fine  adjustment  until  the 
bacteria  come  into  view.  This  focal  point  will  be  passed  without 
noticing  it  if  passed  too  quickly,  if  the  light  is  too  intense,  or  if 
the  light  is  too  dim.  Regulate  the  amount  of  light  by  raising  or 
lowering  the  condenser  and  by  adjusting  the  mirror.  Unstained 
bacteria  are  nearly  colorless  and  appear  as  faint  shadows. 

Bacteria  lose  their  motility  very  quickly  so  if  there  is  a  delay 
of  two  of  three  minutes  in  getting  the  focus  a  wrong  determination 
may  be  made.  In  such  a  case  it  is  better  to  make  a  new  prepara- 
tion. Do  not  make  but  one  preparation  at  a  time. 

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Laboratory  Outline  for  General  Bacteriology 

Bacteria  move  by  means  of  flagella.  These  are  slender,  hair- 
like  processes  usually  several  times  as  long  as  the  bacterial  cell. 
Some  species  have  but  one  on  the  end,  monotrichiate ;  some  have 
a  tuft  on  one  end,  lophotrichiate ;  some  have  them  distributed  all 
over  the  organism,  peritrichiate.  The  arrangement  of  the  flagella 
causes  a  difference  in  the  kind  of  motion.  Special  staining  methods 
are  necessary  in  order  to  see  the  flagella. 

Examine  No.     19  B.  mesentericus 

32  M.  agilis 

"      129  M.  lactis  varians 
20  B.  proteus 
48  B.  pullorum 
23  B.  mycoides 

Examine  preparation  from  a  cake  of  yeast.  Make  drawings 
showing  appearance  of  yeasts,  starch  grains,  and  bacteria. 


GELATIN 

Gelatin  serves  two  purposes.  As  a  solid  culture  medium,  it  is 
a  technical  device  by  which  the  isolation  of  a  single  species  of 
microorganism  is  made  possible.  To  those  organisms  which  secrete 
proteolytic  enzymes  it  serves  as  a  nitrogenous  food  material.  Beef 
extract  and  peptone  are  added  as  food  for  bacteria. 

Gelatin  was  the  first  substance  used  for  a  solid  culture  medium. 
This  medium  was  originated  in  1882  by  Koch  and  has  since  revolu- 
tionized the  science  of  microbiology.  Milk  sugar  and  litmus  are 
also  added  when  acid  production  is  to  be  determined. 

Gelatin  media  will  melt  at  a  temperature  of  about  24°  C  to  26°  C 
solidifying  again  at  20°C  to  a  clear  transparent  jelly. 

Gelatin  plates  do  not  have  to  be  inverted  during  incubation,  as 
do  the  agar  plates,  because  no  water  of  condensation  ordinarily 
collects  on  the  cover  of  the  Petri  dish  in  case  of  the  gelatin. 


AGAR 

Agar  is  a  seaweed  product  used  in  preparing  various  kinds 
of  solid  culture  media.  Ordinarily  agar  is  composed  of  beef  extract, 
peptone  and  agar.  It  becomes  fluid  at  about  85 °C  and  solidifies 
below  40 CC,  becoming  a  very  solid  opaque  jelly,  which  retains  its 
shape  well  in  slants  and  plates. 

Agar  is  commonly  used  in  determining  the  number  of  bacteria 
per  cc.  in  milk,  water,  and  various  other  substances.  A  definite 

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Laboratory  Outline  for  General  Bacteriology 


amount  of  the  material  to  be  tested  is  mixed  with  the  fluid  agar 
and  allowed  to  cool.  Each  bacterium  is  held  in  a  certain  spot  on 
the  agar.  Bacterial  colonies  develop  upon  several  days  incubation 
and  each  colony  represents  one  bacterium  or  clump  of  bacteria 
originally  present. 

Agar  itself  is  not  a  food  for  microorganisms.  Its  special  value 
is  in  hardening  media  used  for  cultivating  organisms  which  grow 
at  a  temperature  above  melting  point  of  gelatin.  This  feature 
has  made  possible  the  great  strides  that  have  been  taken  in  medical 
bacteriology. 


TO  DETERMINE  THE  MELTING  AND  SOLIDIFYING  POINTS 
OF  AGAR  AND  GELATIN 

Place  tubes  of  agar  and  gelatin  in  wire  basket. 
Set  in  pan  with  water  above  level  of  media  in  tubes. 
Insert  thermometer  in  gelatin. 
Place  pan  over  gas  flame  and  watch  thermometer. 
When  liquefied  record  temperature. 

Change  thermometer  to  agar  tube  and  record  melting  temperature. 
Flace  tubes  in  cold  water  and  watch  for  solidifying  point  first  of 
agar,  then  of  gelatin. 

Melting  point  Solidifying  point 

Agar  °C.  °C. 

Gelatin  °C.  °C. 


AIR  ANALYSIS 

Sterile  melted  agar  tubes  will  be  furnished.  Pour  seven  into 
Petri  dishes  using  one  tube  to  a  dish.  In  doing  this  remove  cotton 
stopper  without  touching  end  of  tube.  Raise  one  side  of  the  cover 
of  the  dish  just  enough  to  allow  the  pouring  of  the  agar.  This  is 
done  to  prevent  bacteria  dropping  in  from  the  air.  When  agar  is 
solid  expose  plates  with  cover  removed  for  five  minutes  in  the 
places  designated  below.  Keep  cover  right  side  up  while  off  from 
plate.  The  one  out  of  doors  should  be  taken  away  from  roads  or 
buildings.  Face  the  wind  in  exposing  plate  so  that  bacteria  will 
not  below  from  you  onto  the  plate.  With  red  glass  marking  pencil 
mark  the  plates  with  your  name,  the  date,  and  place  of  exposure. 
After  exposing  plates  place  in  locker  inverted  to  incubate. 

18 


Laboratory  Outline  for  General  Bacteriology 


No.  Bact. 

No.  Molds 

Area  of 
Plates 

Molds 
per  liter 

Bact. 
per  liter 

1  Bacteriology  laboratory 
2  Out  of  doors 

3  Bottling  room  of  dairy 
4  Cow  barn 

5  Dining  room  (at  meal  time) 
6  Study 
7  Control  —  not  exposed 

Counting  Bacteria  and  Molds  on  Air  Plates 

It  has  been  shown  that  the  bacteria  from  ten  liters  of  air  will 
fall  on  one  hundred  square  centimeters  in  five  minutes.  From 
this  is  derived  the  formula 


10  x  no.  bact.  or  molds  on  plate 
area  of  plate 


no.  bact.  or 
=molds  per  liter 
of  air. 


Measure  diameter  of  plate  and 

from  the  following  table  deter 

mine  the  area. 

Diam.  plate 

Area  plate 

86  mm 

58  sq.  cm. 

87 

59 

88 

61 

89 

62 

90 

63.5 

91 

65 

92 

66.5 

93 

68 

94 

69.5 

95 

71 

96 

72.5 

97 

74 

98 

75.5 

99 

77 

100 

78.5 

In  counting  a  plate  place  it  on  the  glass  plate  ruled  in  squares. 
Remove  the  cover  of  the  dish.  Use  the  lens  holding  it  near  the 
eye.  Bring  the  plate  near  enough  to  focus  clearly.  .  Beginning  at 
the  upper  part  of  the  plate  follow  along  the  lines  working  back  and 
forth  until  the  whole  plate  is  counted. 

19 


Laboratory  Outline  for  General  Bacteriology 


From  air  plates  select  seven  bacterial  colonies  of  different  ap- 
pearances and  write  descriptions  of  them  in  note  book.  Then  make 
transfers  from  them  to  agar  slants.  Mark  tubes  so  they  can  be 
identified  with  descriptions.  To  transfer  colony  sterilize  platinum 
loop  by  heating  entire  length  of  wire  to  redness  in  the  gas  flame. 
Pass  .the  lower  part  of  the  handle  three  or  four  times  through  the 
flame.  When  cool  touch  loop  to  the  colony  to  be  transferred. 
Remove  cotton  plug  and  hold  it  so  the  lower  part  will  not  become 
contaminated  by  touching  anything.  Touch  the  loop  to  the  surface 
of  the  agar  at  the  lower  part  of  the  tube  and  make  a  straight 
streak  up  to  the  top.  Care  is  necessary  not  to  cut  down  into  the 
agar.  Replace  cotton  stopper.  Place  tubes  in  locker  to  incubate. 

Glossary  on  pages  57,  58,  59  is  to  be  learned.  These  terms  will 
be  constantly  used  so  it  is  necessary  to  become  familiar  with  them 
now.  There  will  be  a  written  on  it  soon,  probably  at  the  next 
laboratory  period. 


20 


CAUTIONS  AND  DIRECTIONS  FOR  IDENTIFICATION 
OF  UNKNOWN  CULTURES 


Laboratory  Outline  for  General  Bacteriology 

CAUTIONS  AND  DIRECTIONS  FOR  IDENTIFICATION 
OF  UNKNOWN  CULTURES 

J 

1.  Do  keep  cultures  pure  so  correct  results  may  be  obtained. 

Agar  Slants 

2.  Do  touch  only  the  tops  of  cotton  plugs  when  removing  them 
from  and  replacing  them  in  tubes.     While  out  hold  between 
fingers  with  lower  parts  away  from  hand  so  they  will  not  touch 
anything. 

3.  Do  burn  off  cotton  adhering  to  mouth  of  tube  instead  of  pulling 
it  off  with  the  fingers,  rods,  etc. 

4.  Do  sterilize  the  needle  or  loop  by  heating  entire  length  of  wire 
to  redness  and  pass  lower  part  of  holder  through  flame  three 
or  four  times  before  making  a  transfer. 

5.  Do  allow  needle  to  cool  before  using. 

6.  Do  hold  needle  so  nothing  touches  it  while  cooling. 

7.  Do  avoid  opening  tubes  in  currents  of  air.     Bacteria  may  be 
blown  in. 

8.  Do  hold  tube  nearly  horizontal  while  making  transfers   so 
bacteria  cannot  fall  in. 

9.  Do  push  plugs  in  until  they  are  firm. 

10.  Do  use  care  to  keep  all  cultures  pure  even  when  you  think  it  is 
the  last  time  they  will  be  used. 

11.  Do  keep  all  cultures  until  advised  to  discard  them. 

12.  Do  make  records  complete. 

13.  Do  record  all  negative  as  well  as  positive  results. 

14.  Do  remember  that  "chromogenesis"  refers  to  the  color  of  the 
bacterial  growth  itself,  not  to  the  color  of  the  medium  on 
which  the  bacteria  are  growing. 

15.  Do  touch  surface  of  agar  slants  so  lightly  that  needle  or  loop 
never  breaks  into  the  medium.     Bacteria  are  usually  only  on 

the  Surface.  (Continued  on  Page  24) 

\  22 


Laboratory  Outline  for  General  Bacteriology 


CAUTIONS  AND  DIRECTIONS  FOR  IDENTIFICATION 
OF  UNKNOWN  CULTURES 

1.  Don't   allow  cultures  to  become   contaminated.     Later  work 
with  contaminated  cultures  gives  incorrect  results.     The  fol- 
lowing notes  give  some  hints  about  avoiding  contaminations 
and  obtaining  satisfactory  records. 

Agar  Slants 

2.  Don't  lay  cotton  plug  on  the  table  or  let  it  fall  there,  or  hold 
the  inner  part  of  the  plug  next  the  palm  of  the  hand,  or 
allow  it  to  touch  anything.    If  it  accidentally  touches  anything 
burn  off  a  layer  of  the  cotton  before  replacing  plug  in  tube. 

3.  Don't  pull  off  cotton  adhering  to  the  mouth  of  the  tube  with 
the  fingers,  rods,  matches,  etc.    Burn  it  off. 

4.  Don't  use  the  needle  or  loop  without  first  thoroughly  steriliz- 
ing it  by  heating  the  entire  length  to  redness  and  passing  the 
lower  part  of  the  holder  three  or  four  times  through  the  flame. 

5.  Don't  use  the  needle  or  loop  when  hot  enough  to  kill  the 
bacteria. 

6.  Don't  feel  of  the  needle  with  the  fingers  or  touch  it  against 
something  not  sterile  to  see  if  it  is  cool  enough  to  use. 

7.  Don't  open  tubes  in  currents  of  air.    Bacteria  are  blown  in. 

8.  Don't  hold  tubes  vertical  when  open  thus  allowing  bacteria 
to  fall  in. 

9.  Don't  insert  plugs  so  slightly  that  they  fall  out  when  tubes 
are  handled. 

10.  Don't  be  careless  with  cultures  when  you  think  it  may  be  the 
last  time  you  are  going  to  use  them.     You  frequently  find 
they  are  needed  for  later  work. 

11.  Don't  discard  any  cultures  until  you  are  advised  to  do  so. 

12.  Don't  make  partial  records  of  cultures  when  complete  ones 
are  possible. 

13.  Don't  leave  record  spaces  blank  when  you  find  that  no  change 
has  taken  place  or  when  you  obtain  a  negative  reaction. 

14.  Don't  record  the  color  of  the  medium  under  "chromogenesis" 
or  leave  the  space  blank  if  the  bacterial  growth  is  white  or 
colorless. 

15.  Don't  break  the  surface  of  the  agar  slant  with  the  needle  or 
loop.     The  bacteria  are  usually  entirely  on  the  surface  of  the 
agar  and  mixing  them  with  the  agar  greatly  increases  the 
difficulty  of  making  later  inoculations.  (Continued  on  Page  25) 

23 


Laboratory  Outline  for  General  Bacteriology 

16.  Do  take  small  amounts  of  bacteria  for  inoculation.    Only  a  few 
bacteria  are  necessary. 

17.  Do  keep  bacterial  cultures  out  of  the  sunlight,  j 

Liquid  Media 

18.  Do  keep  cultures  upright  enough  so  that  plugs  will  not  become 
wet. 

19.  Same  as  1  to  13  inclusive  for  agar  slants. 

20.  Do  keep  fermentation  tubes  so  no  air  enters  inner  tube. 

21.  Do  sterilize  loop  after  touching  it  to  litmus  paper  in  testing 
for  reaction  before  putting  it  back  into  the  tube  for  another 
loopful. 

22.  Do  handle  incubated  cultures  gently  before  taking  the  ob- 
servations.   This  is  particularly  important  with  nutrient  broth 
as  the  appearance  may  be  greatly  changed  by  jarring. 

23.  Do  use  uninoculated  tubes  of  media  for  comparison  in  making 
observations  and  tests.  • 

Plate  Cultures 

24.  Do  lift  covers  only  high  enough  to  insert  the  pipette  or  needle 
when  adding  inoculation  material. 

25.  Do  have  medium  at  right  temperature  when  pouring — agar 
about  40°  C.,  and  gelatin  between  25°  C.  and  40°  C. 

26.  Do  remove  cotton  plugs  from  tubes  or  flasks  without  touching 
lips  of  tubes  or  flasks  and  burn  off  any  adhering  cotton  that 
would  interfere  with  pouring. 

27.  Do  lift  cover  when  pouring  just  high  enough  so  tube  or  flask 
will  not  scrape  on  edges  of  dish. 

28.  Do  mix  medium  well  with  inoculation  so  bacteria  will  be  evenly 
distributed. 

29.  Do  keep  medium  from  getting  on  edges  of  plate  when  pouring 
or  mixing  as  this  glues  them  together  and  frequently  leads 
to  breakage  and  contamination. 

30.  Do  pour  plates  where  air  is  quiet. 

31.  Do  keep  plates  closed  while  looking  to  see  if  medium  has 
hardened. 

32.  Do  place  plates  near  center  of  table  to  harden  as  edges  of  table 
are  not  level. 

33.  Do  always  carry  gelatin  plates  level  and  right  side  up. 

34.  Do  keep  agar  plates  inverted  after  hardening  and  while  in- 
cubating. (Continued  on  Page  26) 

24 


Laboratory  Outline  for  General  Bacteriology 


16.  Don't  take  all  the  growth  possible  for  the  first  inoculation  as 
you  will  have  about  nineteen  more  to  make  from  the  same 
culture.     Simply  touching  the  cool  sterilized   needle  to  the 
growth  is  usually  sufficient. 

17.  Don't  allow  the  sun  to  shine  on  cultures.     Sunlight  destroys 
certain  species  of  bacteria  very  quickly. 

Liquid  Media 

18.  Don't  lay  tubes  of  liquid  media  on  table  or  hold  them  so  that 
plugs  become  wet.     Contaminations  usually  follow  wet  plugs. 

19.  Same  as  1  to  13  inclusive  for  agar  slants. 

20.  Don't  allow  air  to  get  into  the  small  inner  tube  of  the  fermen- 
tation tubes. 

21.  Don't  put  loop  back  a  second  or  third  time  without  steriliza- 
tion after  touching  it  to  litmus  paper  in  testing  the  reaction. 

22.  Don't  shake  or  handle  tubes  roughly  before  taking  the  obser- 
vations. 

23.  Don't  make  observations  or  tests  of  cultures  without  using 
uninoculated  tubes  for  controls. 

Plate  Cultures 

24.  Don't  lift  covers  clear  off  when  adding  inoculating  material 
with  pipette  or  loop. 

25.  Don't  pour  agar  or  gelatin  when  too  hot  or  too  cold. 

26.  Don't  touch  the  lip  of  the  tube  or  flask  when  removing  cotton 
stopper. 

27.  Don't  scrape  outside  of  tube  or  flask  on  edge  of  plate  when 
pouring. 

28.  Don't  leave  plates  without  mixing  inoculation  with  medium. 

29.  Don't  slop  medium  onto  edges   of  plates   when  pouring  or 
mixing. 

30.  Don't  pour  plates  in  a  current  of  air. 

31.  Don't  remove  or  lift  cover  of  plate  to  see  if  medium   has 
hardened. 

32.  Don't  leave  plates  anywhere  except  near  the  center  of  table 
to  harden  as  tables  are  crowning.     This  makes  the  layer  of 
medium  uneven. 

33.  Don't  ever  carry  gelatin  plates  tipped  or  invert  them  to  in- 
cubate. 

34.  Don't  incubate  agar  plates  right  side  up  as  condensation  water 
causes  trouble  by  dropping  back  from  the  cover, 

(Continued  on  Page  27) 
25 


Laboratory  Outline  for  General  Bacteriology 

35.  Do  allow  agar  to  harden  in  plates  without  tipping  them  around 
and  breaking  the  medium. 

Gelatin  Stab 

36.  Do  have  needle  straight  before  puncturing. 

37.  Do  puncture  clear  to  bottom  of  tube. 

38.  Do  remove  needle  in  line  of  puncture. 

39.  Do  puncture  in  center  of  tube  and  only  once. 

40.  Do  hold  tube  above  medium  so  warmth  of  the  hand  will  not 
melt  the  gelatin. 

41.  Do  watch  cultures  carefully  to  see  when  liquefaction  begins 
and  when  it  is  complete  as  well  as  for  the  form  of  growth 
in  both  liquefying  and  non-liquefying  cultures. 

Morphology 

42.  Do  come  at  right  times  to  make  agar  slants  to  have  them  at 
the  required  age  for  studying  at  laboratory  periods. 

43.  Do  prepare  films  with  only  enough  bacteria  to  show  slight 
cloudiness. 

44.  Do  spread  films  over  the  entire  cover  glass. 

45.  Do  leave  stains  on  the  exact  time  called  for  in  the  directions. 

46.  Do  measure  at  least  ten   bacteria  to  find  the   size   of  the 
majority. 

47.  Do  measure  both  length  and  breadth  of  rod  shaped  bacteria 
and  put  both  measurements  down. 

48.  Do  use  care  to  fill  out  all  points  called  for  under  "Morphology." 

49.  Do  examine  preparations  for  motility  as  soon  as  possible  after 
making,  as  many  bacteria  lose  their  motility  very  quickly ;  and 
do  shut  down  on  the  light  enough  so  bacteria  can  be  seen. 

50.  Do  handle  cover  glasses  with  forceps. 


26 


Laboratory  Outline  for  General  Bacteriology 

35.  Don't  tip  agar  plates  constantly  while  they  are  hardening. 
It  breaks  the  agar  so  it  will  not  form  a  solid  mass  which  can 
be  satisfactorily  inverted. 

Gelatin  Stab 

36.  Don't  puncture  medium  with  a  crooked  needle  or  with  the  loop. 

37.  Don't  stop  puncturing  until  the  needle  touches  the  bottom  of 
the  tube. 

38.  Don't  split  the  medium  when  removing  the  needle. 

39.  Don't  puncture  near  the  side  of  the  tube  or  more  than  once. 

40.  Don't  hold  the  tube  in  the  hand  until  the  warmth  of  the  hand 
melts  the  gelatin. 

41.  Don't  fail  to  record  the  form  of  growth,  when  liquefaction 
begins,  and  when  it  is  complete. 

Morphology 

42.  Don't  forget  to  come  extra  times  when  necessary  to  make 
cultures  so  as  to  have  them  the  right  age  to  use  at  laboratory 
periods. 

43.  Don't  use  too  much  or  too  little  material  in  preparing  films. 

44.  Don't  leave  all  the  material  of  the  film  in  one  small  thick 
mass  which  can  never  be  seen  through.    Spread. 

45.  Don't  leave  stains  on  for  longer  or  shorter  times  than  called 
for  in  the  directions. 

46.  Don't  measure  less  than  ten  different  bacteria  before  filling 
in  measurements  called  for. 

47.  Don't  think  the  size  of  a  rod  simply  means  its  length,  or  that 
you  multiply  the  length  by  the  width. 

48.  Don't  think  that  most  of  the  information  called  for  under 
"Morphology"  does  not  amount  to  much  and  needs  not  to  be 
filled  out. 

49.  Don't  make  preparations  for  motility  examination  until  you 
are  ready  to  examine  them,  or  leave  on  the  full  amount  of 
light  when  trying  to  find  them. 

50.  Don't  ever  touch  the  surface  of  a  cover  glass  with  the  fingers. 
It  leaves  enough  greasiness  to  prevent  the  proper  spreading 
of  films. 


27 


Laboratory  Outline  for  General  Bacteriology 


THE  CHART 

The  purpose  of  the  descriptive  chart  is  to  provide  a  uniform 
and  concise  method  of  recording  nearly  all  the  ordinary  and  some 
of  the  unusual  observations  concerning  the  morphology  and  activi- 
ties of  bacteria.  It  should  familiarize  students  with  bacterial  char- 
acteristics and  facilitate  in  the  grouping  and  identification  of  species. 

For  preliminary  practice  in  handling  bacteria  five  pure  cultures 
will  be  given  each  student.  This  work  includes  the  Gram's  stain, 
fuchsin  stain  for  measurement,  inoculations  into  and  observations 
on  agar  slants,  gelatin  stabs,  nutrient  broth,  milk,  litmus  milk, 
potato,  and  the  nitrite  test. 

After  this  six  unknown  cultures  are  given  each  student.  All 
data  called  for  on  the  Descriptive  Chart  is  worked  out  and  filled  in. 
When  complete  the  cultures  are  named  by  comparing  descriptions 
with  those  on  pages  43,  44,  45,  46.  For  work  on  unknown  cultures 
12-14  laboratory  periods  are  given. 

No  bacteria  pathogenic  to  man  are  given  out  to  students  in 
this  course. 


28 


Laboratory  Outline  for  General  Bacteriology 


DESCRIPTIVE  CHART,  ENDORSED  BY  SOCIETY  OF  AMERICAN   BAG 
TERIOLOGISTS  AT  THE  ANNUAL  MEETING  DEC.  30,   1920. 

Prepared  by  H.  J.  Conn,  K.  N.  Atkins,  I.  J.  Kligler,  J.  F.  Norton,  G.  E. 
Harmon,    Committee   on    bacteriological   technic. 

MORPHOLOGY 

Note.  Underscore  required  terms. 
Vegetative    Cells,    Medium    used.. 


temp   age days. 

Form,  spheres,  short  rods,  long  rods,  filaments,  commas,  short  spirals,  long 
spirals,  curved. 

Arrangements,  single,  pairs,  chains,  fours,  clusters,  cubical  packets. 

Limits  of  Size  Size  of  Majority  

Ends,  rounded,  truncate,  concave. 
Capsules,  present  on  

How    stained    

Sporangia,  present,  absent.     Medium  used 

temp  age days. 

Form,  elliptical,  short  rods,  spindled,  clavate,  drumsticks. 

Limits  of  Size  Size  of  Majority  

Endospores,  present,  absent. 

Location  of  endospores,  central,  polar. 

Form,  spherical,  elliptical,   elongated. 

Limits  of  Size  Size  of  Majority  

Wall,  thick,  thin. 

Sporangium  wall,  adherent,  not  adherent. 

Motility 

In    broth    On    agar 

Flagella.  No Attachment,  polar,  bipolar  peritrichiate. 

How  stained  

Irregular  Forms. 

Present  on   in days   at    °C. 

Form  spindled,  cuneate,  filamentous,  branched, 

or    

Staining   Reactions 

Gram   Acid  fast   

Special   Stains   


CULTURAL  CHARACTERISTICS 

Underscore  required  terms. 

Agar  Stroke.     Incubation  temp °C,  Age days 

Growth,  scanty,  moderate,  abundant,  none. 

Form   of  growth,   filiform,     echinulate,     beaded,     spreading,     arborescent, 

rhizoid. 

Elevation  of  growth,  flat,  effuse,  raised,  convex. 
Lustre,  glistening,  dull. 
Topography,  smooth,  contoured,  rugose. 

Optical  Characters,  opaque,  translucent,  opalescent,  iridescent. 
Chromogensis  Photogenic.     Fluorescent. 

29 


Laboratory  Outline  for  General  Bacteriology 


Odor,    absent,    decided,    resembling    

Consistency,  butyrous,  viscid,  membranous,  brittle. 
Medium,  grayed,  browned,  reddened,  blued,  greened. 

Gelatin  Stab,  Incubation  temp °C,  Age  days 

Growth,  uniform,  best  at  top,  best  at  bottom. 

Line  of  puncture,  filiform,  beaded,  papillate,  villpus,  arborescent. 
Liquefaction,  none,  crateriform,  napiform,  infundibuliform,  saccate,  strati- 
form, begins  in d  complete  in d. 

Depth  of  liquefaction  in  tube  of  10   mm.  diameter  evenly  inoculated   at 

20°C  for  30  days   mm. 

Medium,  fluorescent,  browned. 

Potato,  Incubation  temp °C,  Age  days 

Terms  as  in  Agar  stroke. 

Nutrient  Broth,   Temp  °C,  age  days 

Surface  growth,  ring,  pellicle,  flocculent  membranous,  none. 
Clouding,  slight,  moderate,  strong,  transient,  persistent,  none,  fluid  turbid. 
Sediment,  compact,  flocculent,  granular,  flaky,  viscid  on  agitation,  abun- 
dant, scant,  none. 

Agar  Colonies,  temp  °C,  Age  days 

Growth,  slow,  rapid. 

Form,  punctiform,  circular,  irregular,  mycelioid,  filamentous,  rhizoid. 
Surface,  smooth,  rough,  concentrically  ringed,  radiate. 
Elevation,  flat,  effuse,  raised,  convex,  pulvinate,  umbonate. 
Edge,  entire,  undulate,lob  ate,  erose,  filamentous,   curled. 
Internal    structure,    amorphous,    finely-,    coarsely-,    granular,    filamentous, 
curled,  concentric. 

Gelatin  Colonies,  temp  °C,  Age  days 

Growth,   slow,  rapid. 

Form,  punctiform,  circular,  irregular,  mycelioid,  filamentous. 
Elevation,  flat,  raised,  convex,  pulvinate,  crateriform,  (liquefying) 
Edge,  entire,  undulate,  lobate,  erose,  filamentous,  floccose,  curled. 
Liquefaction,  cup,  saucer,  spreading. 

Internal    structure,    amorphous,    finely-,    coarsely-,    granular,    filamentous, 
curled,  concentric. 

PHYSIOLOGY 

TEMPERATURE    RELATIONS 

Optimum  temperature  for  growth °C. 

Maximum    temperature    for    growth °C 

Minimum  temperature  for  growth °C. 

CHROMOGENESIS 

Nutrient  broth 

Nutrient    gelatin 

Nutrient    agar 

Potato... 


PRODUCTION   OF   INDOL 

Medium : 

Indol    absent,   present   in days 

PRODUCTION    OF    HYDROGEN    SULFIDE 

Medium : 

H2S   absent,   present    in days 

30 


Laboratory  Outline  for  General  Bacteriology 


FERMENTATION 

Temperature. 


Medium 
containing     

w 

u 

CO 

o 

H 

W 

o 

M 

< 

2 

s 

g 

C3 

H 

1 

H 
£ 

and: 

< 
j 

H 

5 

< 
w 

CJ 

Gas 

First  appearance  of  acid  

First  appearance  of  alkali  .   .            .   . 

Reaction  after                                   days 

Reaction  after                                   days 

Max.  H-ion  Cone.  . 

RELATION   TO   OXYGEN 

Method    used 

Medium     Temperature °C. 

Aerobic    growth ;    absent    present,    better    than    anaerobic    growth. 

Anaerobic    growth:    absent,   occurs    in    presence    of   dextrose,    of   sucrose,    of    lactose,    of    nitrate; 
better     than     aerobic    growth. 


DIASTATIC   ACTION 

Breadth  of  clear  zone  on  starch  agar  plates, 
in days  : 


MILK 

Temperature.  . 

Reaction:  1  day 2  days 4  days 7  days. 

Acid   curd:  1  day 2  days 4  days 7  days. 

Rennet   curd:  1  day 2  days 4  days 7   days. 

Peptonizat  on  :  1  day 2  days 4  days 7   days. 

Reduction    of    litmus    in days;    of  methylene   blue    in days. 

Influence     of     indicator     on     growth .  .  


10  days. 

... .10  days. 

10  days. 

10  days. 


Nitrite 
Gas: 


Gas: 
Nitrite 


NITRATE    REDUCTION 

Medium Temperature °C. 

1  day 2  days 4  days 7  days 10  days 

1  day 2  days 4  days 7  days 10  days 

Medium Temperature °C. 

1  day 2  days 4  days 7  days 10  days 

1  day 2  days .  .4  days 7  days 10  days 


Index    No.* 

BRIEF  CHARACTERIZATION 

As  each  of  the  following  characteristics  is  determined,  indicate  in  proper  marginal  square  by 
means  of  figure,  as  designated  below: 


PRIMARY  CHARACTERISTICS 

Microscopic 
Features 

Form:      1.  streptococci;     2,  diplococci;    3,    micrococci;     4,   sarcinae;     5,  rods; 
6,  commas;    7,  spirals;   8,  branched  rods;  9,    filamentous 

Spores:     1.  central;    2,  polar;    3,  absent 



Flagella:     1,  peritrichic;    2,  polar;    3,  absent 

Gram  stain:     1,  positive;   2,  negative 

Miscellaneous 
Biochemical 
Reactions 

Pathogenicity,  etc.:     1,   for   man;    2,  for    animals;    3,  for  plants;    4,  parasitic 
but  not  pathogenic;   5,  saprophytic;   6,  autotrophic 

Relation  to  oxygen:     1,  strict  aerobe;    2,  facultative  anaerobe;   3.  strict  aerobe 

Gelatin  liquefaction:     1,  positive;   2,  negative 



In  nitrate  media:     1,  nitrite  and  gas;    2,  nitrite  but  no  gas;    3,  neither   nitrite 
nor  gas 

Chromogenesis:      1,  flourescent;      2.  violet;      3.  blue;      4,  green;      5,  yellow; 
G,  orange;    7,  red;    8,  brown;    9,  pink;    0.  none 

Carbohydrate 
Reactions 

Diastatic  action:      1.  positive;  2,  negative 



From  dextrose:     1,  acid  and  gas;   2,  acid  without  gas;   3.  no  acid 

From  lactose:     1.  acid  and  gas;    2,  acid  without  gas;    3.  no  acid 

From  sucrose:     1,  acid  and  gas;    2,  acid  without  gas;    3,  no  acid 

SECONDARY  CHARACTERISTICS 

Vegetative 
Cells 

Diameter:     1,  under  0.5M;    2,  between  0.5A  and  1H\   3,  over  1M 

Length  :     1,  less  than  2  diameters;   2.  more  than  2  diameters 



Chains  (4  or  more  cells):     1.  present;    2,  absent 

Capsules:     1.  present;    2,  absent 

in 
o> 

i 

Shape:     1,  round;    2,  oval  to  cylindrical 

Diameter:     1,  less  thin  diameter  of  rod;    2,  greater  than  diameter  of  rod 

Cultural  Features 

Agar  Stroke 

Abundance:     1.  abundant;    2,  moderate;  ^3.  slight;   4,  absent 

Lustre:     1,  glistening;    2,  dull 

Surface:     1,  smooth;    2,  contoured;    3,  rugose 

Agar  colonies:       1,  panctiform;      2,  round  (over  1  mm.  diameter);      3,  rhizoid; 
4,  filamentous;    5,  curled 

Gelatin  colonies:    I.  punctiform;    2.  round  (over  1  mm.);    3,  irregular;   4.  fila- 
mentous 

M 

1 

Acid:     1,  sufficient  for  curdling;   2.  insufficient  for  curdling;   3,  no  acid 

Rennet  curd:     1,  present;    2.  absent 

Peptonizatioo,:      1,  present;    2,  absent 

*  Recording  the  "Index  Number"  here  is  optional  ;  but  its  use  will  be  found 
convenient  if  the  charts  are  to  be  filed  according  to  the  salisnt  characteristics  of  the 
organisms.  The  Index  Number  consists  of  the  firtt  thirteen  figures  from  the  margin 
(primary  characteristics)  copied  down  in  the  order  of  their  occurrence  in  the  margin, 
placing  a  dash  wherever  a  heavy  rule  occurs  in  the  margin.  Thus,  B.  coli  belongs  to 
the  group  5312-41220-1111. 


PLATE.     1. 


0. 


c. 


J 

v^ 


A.      Stab    Cultures 

1.  Filiform 

2.  Beaded 

3.  Villous 

4.  Arborescent 

5.  Papillate 


B.      Type   of   Liquefaction      C. 


Stratiform 
Napiform 
Infundibuliform 
Crateriform 


5.  Saccate 


Streak  Cultures 

1.  Arborescent 

2.  Spreading 

3.  Filiform 

4.  Beaded 

5.  Echinulate 


33 


Laboratory  Outline  for  General  Bacteriology 


AGE  OF  CULTURES,  TO  DETERMINE 


Motility 

Flagella 

Spores  and  sporangia 

Stains  for  measurement  of  vegetative  cells 

Gram's  stain 


24  hours 

24       " 

3  days  to  1  week. 

24  hours 

24       " 


AGE  OF  VARIOUS  CULTURES  WHEN  EXAMINED 


Agar  slants 
Potato  slants 
Nutrient  broth 
Gelatin  stabs 

Agar  plates 
Gelatin  plates 
Dunham's  peptone 
for  Indol  test 
Lead  acetate  agar 
for  H0S  test. 
Milk  " 
Litmus  milk 
Nitrate  broth 
Nitrate  agar 
Starch  agar  for 
diastatic  action 

Fermentation 
tubes  of  dextrose 
lactose,  sacch- 
arose, glycerine 


described  after  7  days,  checked  up  at  14  days 

same  as  agar  slants 

same  as  agar  slants 

examine  every  laboratory  period  until  30  days 

old,  recording  any  changes 
examine  after  1  week 
from  2  to  7  days 

4  days 

7  days 

1,  2,  4,  7,  and  10  days 
1,  2,  4,  7,  and  10  days 
1,  2,  4,  7,  and  10  days 
1,  2,  4,  7,  arid  10  days 

7  days,  unless  the  growth  is  very  rapid,  then 
the  test  is  made  sooner. 


examined  2,  7  and  14  days. 


TEMPERATURE  OF  INCUBATION 

All  cultures  are  to  be  grown  at  locker  temperature,  which  is 
about  20°  C.  except  the  one  set  of  agar  slants  made  for  testing 
growth  at  37°  C. 


34 


Laboratory  Outline  for  General  Bacteriology 

STAINS 

Their  Composition  and  Methods  of  Use 

Standard  Alcoholic  Solutions.  Standard  alcoholic  solutions  of 
anilin  colors,  especially  fuchsin,  gentian  violet,  and  methylene  blue, 
are  made  by  dissolving  10  grams  of  dry  color  in  100  cc  of  95% 
alcohol.  These  are  used  as  stock  solutions  from  which  the  various 
stains  are  made. 

Watery  Fuchsin.    Made  by  diluting  one  part  of  standard  alco- 
holic solution  of  fuchsin  with  nine  parts  of  distilled  water. 
Use.    Leave  on  1  minute,  wash  with  water. 

Watery  Gentian  Violet.  Made  by  diluting  one  part  of  standard 
alcoholic  solution  of  gentian  violet  with  nine  parts  of  distilled 
water. 

Use.    Leave  on  1  minute,  wash  with  water. 

Carbol  Fuchsin.  Made  by  diluting  one  part  of  standard  alco- 
holic solution  of  fuchsin  with  nine  parts  of  a  five  per  cent,  solution 
of  carbolic  acid. 

Use.    Leave  on  1/2  minute,  wash  with  water. 

Methylene  blue,  Loeffler's.  Made  by  adding  thirty  cc  of  stand- 
ard alcoholic  solution  of  methylene  blue  to  seventy  cc  of  potassium 
hydroxide  solution  (1-10,000). 

Use.    Leave  on  10  minutes,  wash  with  water. 

Gram's  stain  (Chester).  Made  by  shaking  1  cc  of  aniline  oil 
with  10-15  cc  of  distilled  water  vigorously  for  several  minutes  and 
filtering.  To  10  cc  of  the  filtrate  add  1  cc  of  standard  alcoholic 
solution  of  gentian  violet  and  filter  again.  This  is  the  stain.  The 
Gram's  solution  has  the  following  composition: 
Metallic  iodine  1  gram 

Potassium  iodide  2  grams 

Water,  distilled  300  cc 

Use.  Anilin  gentian  violet  Vr>  minute,  wash  with  water 

Gram's  mixture  (iodine) 
Alcohol  95 ',  about      1 

Flagella  stain  (Duckwall).    Mordant  is  prepared  as  follows: 
Desiccated  tannic  acid  2  grams 

Water,  distilled  15  cc 

Saturated  alcoholic  solution 

of  fuchsin  1  cc 

Normal  NaOH  solution  2-4  drops 

35 


Laboratory  Outline  for  General  Bacteriology 

When  ready  to  use  mix  with 
Ferrous  sulphate,  saturated 

aqueous  solution  5  cc 

Filter. 

Make  stain  by  adding  2  cc  of  saturated  alcoholic  solution  of 
fuchsin  to  8  cc  of  a  5%  solution  of  carbolic  acid. 

Use  Mordant  V$> — 1  minute,  wash  with  water. 

Carbol    fuchsin    1/2 — 1  minute,  wash  with  water. 


MORPHOLOGY 

The  size,  form,  arrangement,  etc.,  of  vegetative  cells  is  deter- 
mined from  twenty-four  hour  old  agar  slant  cultures.  Watery 
fuchsin  stain  is  used  as  it  has  been  found  satisfactory  with  most 
bacteria. 

The  Gram  stain  is  made  at  the  same  time.  This  stain  is  for 
differential  purposes  only.  Some  bacteria  keep  the  stain  and  some 
do  not.  When  the  bacteria  remain  dark  blue  they  are  recorded 
as  Gram  positive,  otherwise  as  Gram  negative. 

Preparation  of  films.  The  method  of  preparing  films  for  these 
two  stains  is  as  follows : 

Place  cover  glasses  in  forceps  without  touching  them  with  the 
fingers. 

With  sterile  loop  place  a  drop  of  water  on  each  of  two  cover 
glasses. 

With  a  cool  sterile  needle  or  loop  transfer  from  edge  of  culture 
in  lower  part  of  tube  to  the  drops  of  water  on  the  cover  glasses. 
Use  only  enough  to  cloud  the  water  slightly.  Burn  any  excess 
of  bacteria  from  the  loop  and  spread  the  drops  over  the  surface  of 
the  cover  glasses.  If  a  drop  will  not  remain  spread  but  rolls 
together  again  it  shows  that  the  cover  glass  is  greasy  and  should 
be  discarded. 

Air  dry  without  heat. 

Fix  film  to  the  cover  glass  by  passing  it  three  times  through 
the  gas  flame.  Keep  the  film  side  up.  Do  not  hold  in  flame  longer 
than  you  could  hold  your  finger  in  it  without  burning. 

They  are  now  ready  to  be  stained.  For  staining  methods  see 
page  35, 

After  staining  drying  may  be  hastened  by  blotting  with  filter 
paper.  When  dry  mount  on  slide  film  side  down  in  drop  of  cedar 
oil. 

Label  slides  carefully. 

Never  pile  slides  together.    Place  them  in  slide  boxes. 

36 


Laboratory  Outline  for  General  Bacteriology 


Motility  and  flagella  staining.  Motility  is  determined  from 
day-old  nutrient  broth  or  agar  cultures.  Motile  cultures  are  stained 
for  flagella.  Preparations  are  made  as  follows:  With  a  pipette 
place  about  4  drops  of  water  together  in  a  watch  glass.  Inoculate 
center  of  water  with  culture  taken  from  edge  of  growth  in  lower 
part  of  agar  tube.  Be  sure  the  needle  is  cool  before  starting  and 
avoid  any  rapid  vigorous  movements  as  the  flagella  are  easily 
broken  off.  Use  enough  bacteria  to  cloud  the  center  of  the  water 
slightly.  Let  the  dilution  stand  for  five  minutes,  then  remove  from 
edge  of  dilution  several  loopfuls  onto  clean  cover  glasses.  Do  not 
spread  the  loopfuls  but  let  them  dry  on  the  several  spots  where 
they  were  placed.  The  best  flagella  are  usually  found  near  the 
edges  of  the  drops.  Air  dry,  and  pass  once  through  the  flame. 
Cover  with  freshly  mixed  mordant  \/*>  to  1  minute.  Wash  and  stain 
with  carbol  f  uchsin  1/2  to  1  minute.  Wash,  dry,  and  mount. 

Sporangia  and  endospores.  Determinations  on  these  are 
usually  made  from  watery  fuchsin  stains  from  week-old  cultures. 
Sometimes,  however,  it  is  necessary  to  use  younger  cultures  in  order 
to  find  sporangia. 

AGAR  STROKE 

Agar  medium  is  placed  in  test  tubes  and  cooled  in  a  slanting 
position.  This  leaves  a  large  surface.  Inoculations  are  made  in 
one  streak  the  entire  length  of  the  slanted  surface,  care  being 
exercised  not  to  break  down  into  the  medium.  For  comparison 
of  growth  at  different  temperatures  two  tubes  are  inoculated  from 
each  culture.  These  are  kept  at  20 °C.  and  37 °C.  respectively.  De- 
scriptions are  made  at  7  and  14  days. 

GELATIN  STAB 

Gelatin  is  used  in  tubes  of  about  10  mm.  diameter.  The  depth 
is  about  4  cm.  Inoculation  is  made  with  a  straight  needle  stabbing 
once  in  the  center  to  the  bottom  of  the  tube.  They  are  watched 
to  determine  when  liquefaction  begins  and  is  complete  as  well  as 
for  the  form  of  both  liquefiers  and  non-liquefiers.  Cultures  are  kept 
30  days. 

POTATO  CULTURES 

Potatoes  cut  in  cylindrical  pieces  with  a  slanted  surface  are 
sterilized  in  tubes  having  moistened  cotton  on  the  bottom  to  prevent 
drying  out  of  the  potato.  Inoculation  is  made  in  a  streak  up  the 
slanted  surface.  After  incubation  description  is  made  in  the  same 
manner  and  using  the  same  terms  as  for  agar  slants.  Records 

37 


Laboratory  Outline  for  General  Bacteriology 


are  made  on  the  chart  in  the  blank  space  with  heading  "Medium 
solid." 

NUTRIENT  BROTH 

Nutrient  broth  is  made  from  beef  extract,  peptone,  and  distilled 
water.  After  inoculation  the  nutrient  broth  cultures  must  be 
handled  carefully  to  avoid  shaking  down  any  pellicle  or  surface 
growth  that  may  be  present.  After  recording  surface  growth  and 
clouding,  which  may  be  distinguished  better  by  comparison  with 
an  uninoculated  tube,  the  character  of  the  sediment  may  be  deter- 
mined by  giving  the  tube  a  whirling  motion  and  observing. 

AGAR  PLATES  FOR  COLONY  DESCRIPTIONS 
AND  SUNLIGHT  TEST 

Inoculate  a  water  blank  with  a  small  amount  of  culture  taken 
from  the  original  agar  slants.  Shake,  and  with  a  sterile  loop  trans- 
fer once  from  water  blank  to  a  tube  of  melted  agar.  Shake  agar 
without  having  medium  touch  the  cotton  plug.  Sterilize  loop  and 
make  two  transfers  from  first  agar  tube  to  a  second.  Pour  both 
agar  tubes  into  plates  marking  them  "a"  and  "b"  in  addition  to 
the  culture  number.  When  cool  paste  a  thick  opaque  paper  over 
one-half  the  bottom  of  each  plate.  This  is  to  shut  off  the  light. 
Expose  plates  to  the  sunlight  for  fifteen  minutes,  watching  to  see 
no  shadows  creep  over  them.  Place  in  locker  to  incubate.  After 
seven  days  make  descriptions  of  both  surface  and  deep  colonies 
from  the  plate  that  shows  the  most  favorable  number  of  bacteria. 

To  determine  the  percentage  of  bacteria  killed  by  sunlight 
count  an  equal  area  on  both  the  shaded  and  sun  exposed  portion 
of  the  Petri  dish.  Subtract  the  number  of  bacteria  in  sun  exposed 
area  from  the  number  in  the  shaded  area  and  divide  by  the  number 
of  bacteria  in  the  shaded  area. 

Example.  Number  of  bacteria  in  shaded  area  28,  number  in 
sun  exposed  are  12.  28—12=16  16-28=57.1  The  result  is 
approximately  57  per  cent. 

GELATIN  PLATES 

These  are  made  in  the  same  manner  as  the  agar  plates  but 
are  not  exposed  to  sunlight.  Incubate  in  the  locker  right  side  up. 
Liquefying  bacteria  must  be  watched  carefully  to  get  descriptions 
before  the  gelatin  is  entirely  liquefied. 

NITRATE  BROTH 

Nitrate  broth  is  a  medium  made  by  using  small  amounts  of 
peptone  and  potassium  nitrate  in  tap  water.  After  cultures  have 

38 


Laboratory  Outline  for  General  Bacteriology 


grown  for  the  required  length  of  time  tests  are  made  as  follows: 
With  sterile  pipette  remove  2  cc.  of  culture  to  a  clean  test  tube.  Add 
1  cc.  of  test  solution  which  is  made  up  of  naphthylamin,  acetic  acid, 
sulphanilic  acid  and  water.  A  pink  color  shows  the  presence  of 
nitrite.  Test  an  uninoculated  tube  in  the  same  manner  for  a  con- 
trol. Observations  are  made  for  gas  bubbles  rising  in  the  nitrate 
broth  cultures. 

NITRATE  AGAR 

Nitrate  agar  is  the  same  as  standard  agar  with  .1  per  cent 
KNO .  added.  It  is  used  in  slants,  which  are  inoculated  by  making 
both  a  streak  on  the  surface  and  a  stab  to  the  bottom  of  tube.  The 
test  solution  is  poured  over  the  surface  of  the  medium.  Gas  pro- 
duction is  shown  by  cracks  in  the  agar. 

HYDROGEN  SULPHIDE 

A  beef  extract  agar  very  rich  in  peptone  and  containing  lead 
acetate  is  used  in  tubes.  This  is  inoculated  by  stabbing  with  a 
straight  needle.  A  darkening  of  the  medium  within  a  few  days 
shows  that  hydrogen  sulfide  has  been  produced. 

INDOL  PRODUCTION 

Cultures  are  grown  in  Dunham's  peptone  solution.  It  is  com- 
posed of  peptone,  sodium  chloride  and  distilled  water.  After  four 
days  the  Ehrlich  test  is  made  as  follows:  Add  to  the  culture  1  cc. 
of  a  2'  '<  solution  of  paradimethylaminobenzaldehyde  in  95 '  <  alcohol. 
Then  add  drop  by  drop  y»  cc.  of  concentrated  HC1.  If  indol  is  pres- 
ent a  pink  zone  appears  which  deepens  and  widens  on  standing. 
The  test  may  be  confirmed  by  shaking  the  culture  with  chloroform 
to  see  if  the  pigment  dissolves.  If  it  proves  soluble  the  test  is 
considered  positive. 

FERMENTATION  TUBES 

The  medium  is  nutrient  broth  with  one  per  cent  of  the  different 
sugars  added.  It  is  put  into  tubes  in  which  are  small  inverted 
inner  tubes  which  are  also  filled  with  the  medium.  If  gas  is  formed 
some  of  it  will  collect  in  the  inner  tube  and  the  percentage  can 
be  measured  by  the  gasometer  chart  shown  on  page  41.  If  the 
bacteria  grow  only  within  the  inner  tube  (this  is  shown  by  cloudi- 
ness or  sediment)  it  shows  that  they  can  grow  only  in  the  absence 
of  oxygen  and  are  anaerobic.  When  growth  occurs  only  outside 
the  small  tube  it  shows  that  the  bacteria  require  oxygen  and  are 
aerobic.  When  growth  occurs  both  inside  and  outside  the  inner 

39 


Laboratory  Outline  for  General  Bacteriology 

tube  it  is  called  facultative  anaerobic.  Observations  on  growth 
and  tests  for  alkalinity  or  acidity  are  made  usually  at  2,  7,  and  14 
days. 

STARCH  AGAR 

Starch  agar  is  like  the  ordinary  agar  with  .2(/<  of  soluble  starch 
added.  It  may  be  poured  hot  into  plates.  When  cool  it  is  inocu- 
lated by  making  one  streak  across  the  center.  Do  not  let  the  loop 
cut  down  into  the  agar.  Invert  to  incubate. 

After  the  culture  has  grown  the  required  length  of  time,  usually 
7  days,  it  is  covered  with  a  solution  of  iodine.  If  the  starch  is 
unchanged  by  the  bacteria  the  entire  plate  will  turn  blue.  When 
diastase  is  present  a  clear  zone  will  appear  along  the  line  of  inocu- 
lation. The  width  of  the  clear  zone  shows  the  intensity  of  the 
diastatic  action.  This  is  measured  in  millimeters  from  the  edge 
of  the  growth  to  the  edge  of  the  blue.  Records  should  be  made 
at  once  as  the  blue  reaction  disappears  within  a  few  minutes. 

MILK  AND  LITMUS  MILK 

Fresh  skimmed  milk  is  tubed  and  sterilized.  For  the  litmus 
milk  enough  litmus  solution  is  added  to  give  it  a  distinctly  blue 
color. 

In  testing  reaction  of  milk  sterilize  loop  and  take  a  loopful 
out  onto  litmus  paper.  If  it  is  necessary  to  take  a  second  loopful 
sterilize  loop  before  taking  it  to  avoid  contaminating  the  culture. 
Test  an  uninoculated  tube  at  the  same  time  for  comparison.  Fresh 
milk  has  what  is  called  an  amphoteric  reaction,  that  is,  it  turns 
red  litmus  slightly  blue  and  blue  litmus  slightly  red.  If  bacteria 
have  produced  a  change  of  reaction  litmus  paper  will  be  turned 
redder  or  bluer  than  the  control.  Milk  and  litmus  milk  tubes  from 
the  same  culture  should  show  the  same  reaction. 

Coagulation  is  when  milk  becomes  thick  like  sour  milk.  Pep- 
tonizatiori  is  shown  by  a  clearing  or  whey  like  appearance  due  to 
destruction  of  the  casein.  It  generally  begins  at  the  surface  and 
works  downward.  The  milk  may  or  may  not  be  curdled.  Sometimes 
when  a  hard  curd  is  formed  whey  is  pressed  out.  This  is  called 
"extrusion  of  whey"  and  can  usually  be  distinguished  from  pep- 
tonization  by  the  clearness  of  the  whey.  Reduction  refers  to  the 
destruction  of  litmus  color.  When  complete  the  litmus  milk  looks 
like  the  plain  milk.  Reduction  usually  begins  at  the  bottom  of  the 
tube  and  works  upward.  Sometimes  the  color  becomes  pale  through- 
out the  entire  tube.  A  change  of  color  to  red  or  blue  indicates 
change  of  reaction  not  reduction. 

40 


Laboratory  Outline  for  General  Bacteriology 


THERMAL  DEATH  POINT 

The  thermal  death  point  of  an  organism  is  customarily  defined 
as  the  least  temperature  that  will  destroy  it  in  ten  minutes  under 
known  conditions.  Several  factors  influence  the  thermal  death 
point.  Old  cultures  are  less  resistant  than  younger  ones.  An  acid 
medium  renders  heat  much  more  effective.  Moist  heat  is  much 
more  efficient  than  dry  heat.  For  comparative  work  it  is  necessary 
to  use  media  of  uniform  reaction  and  composition.  The  presence 
of  spores  indicates  that  the  organism  has  two  thermal  death  points ; 
one  for  the  spores  and  another  lower  one  for  the  vegetative  cells. 
The  medium  recommended  by  the  Committee  on  Identification  of 
Species  of  the  Society  of  American  Bacteriologists,  is  nutrient 
broth.  Several  tubes  are  inoculated  then  heated  in  water  baths 
at  different  temperatures  ranging  usually  from  50  °C  to  70  °C  for 
10  minutes.  They  are  then  incubated  to  see  if  growth  takes  place. 


Fermentation  Tube  Chart 

PLATE     2. 


Laboratory  Outline  for  General  Bacteriology 


DESCRIPTIONS  OF  VARIOUS  SPECIES  OF  BACTERIA 

The  need  has  long  been  felt  of  having  for  student  use  a  variety 
of  bacterial  cultures  that  have  been  so  carefully  worked  through 
that  their  characteristics  are  known.  With  this  in  view  named 
cultures  have  been  obtained  from  the  American  Museum  of  Natural 
History  in  New  York  and  from  various  other  laboratories  as  well 
as  from  our  own.  '  These  have  been  carefully  worked  through,  in 
most  cases  several  timesr  and  by  different  persons.  Work  on  doubt- 
ful points  has  been  repeated  many  times. 

Anyone  familiar  with  similar  work  will  understand  the  diffi- 
culties encountered,  such  as  changes  in  the  cultures  due  to  pro- 
longed cultivation  on  artificial  media,  differences  in  appearances  of 
cultures  and  in  reactions  due  to  differences  in  materials,  especially 
peptone,  from  which  the  culture  media  are  made,  differences  due 
to  personal  equation,  etc. 

We  have  retained  the  names  of  bacteria  as  they  were  sent  to 
us  even  when  in  a  few  cases  it  is  believed  they  were  named  incor- 
rectly. 

In  the  condensed  form  in  which  the  descriptions  are  tabulated 
we  have  not  found  it  practical  to  always  give  all  the  characteristic 
points  and  details  in  regard  to  a  culture.  The  main  points,  how- 
ever, are  given  in  the  majority  of  cases. 


The  rii      ire  In  the  ina*. 
Number,  wnioh  refers  to  pathogen! city, 
has  been  omitted  from  these  charts. 


42 


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V)            IO 

IO    IO    «O    «O    «0    to 

Part  II 


WATER  TESTING 

The  samples  of  water  to  be  tested  are  from  wells,  springs,  res- 
ervoir, and  swimming  pool.  Two  kinds  of  media  are  used — agar  for 
plate  cultures  to  give  the  bacterial  count,  and  a  bile  salts  medium 
in  fermentation  tubes  to  show  gas  production.  The  bile  salts  in- 
hibit the  growth  of  nearly  all  bacteria  other  than  the  intestinal 
forms.  More  than  10  '<  of  gas  in  the  tubes  after  incubating  two 
days  at  37°  C.  is  regarded  as  a  positive  presumptive  test  for  B.  coli 
and  the  water  is  considered  unsafe  for. drinking  purposes.  When 
confirmation  i?  desired  further  tests  are  necessary. 

No  absolute  standard  can  be  given  to  determine  the  potable 
quality  of  water  from  the  number  of  bacteria  in  it.  Water  con- 
taining thousands  of  germs  to  the  cubic  centimeter  may  be  far 
less  dangerous  than  one  containing  but  two  germs  if  one  of  these 
two  be  a  typhoid  bacillus.  It  is  not  the  number  that  proves  dan- 
gerous, it  is  the  kind.  The  agar  count  at  37°  C.  eliminates  the 
water  flora  but  not  the  soil  bacteria.  Harrison  states  that  for  deep 
waters  the  agar  count  should  generally  -not  exceed  10  per  cc.  and 
for  surface  water  not  over  100  per  cc. 

Four  agar  plates  are  made  from  each  sample  using  1  cc.,  .5cc., 
.2  cc.,  and  .1  cc.  respectively  from  the  sample  of  water  which  has 
been  shaken  vigorously  twenty-five  times.  Five  bile  salts  fermen- 
tation tubes  are  inoculated  with  1  cc.  each. 

The  work  should  be  done  in  the  following  order. 

Make  records  in  note  book  giving  each  sample  an  experiment 
number  and  one  page. 

Mark  Petri  dishes  and  tubes  with  locker  number,  sample 
number,  and  amount  of  water  added. 

With  pipettes  graduated  to  tenths  of  a  cubic  centimeter  add 
the  required  amounts  of  water  to  the  plates  and  fermentation 
tubes.  These  pipettes  have  eleven  marks  or  graduations.  They 
are  marked  beginning  at  the  top  o,  .1,  .2,  .3,  .4,  .5,  .6,  .7,  .8,  .9,  and 
1.  Use  the  forefinger,  never  the  thumb,  on  the  top  of  the  pipette 
to  control  the  flow  of  water  from  the  pipette.  When  1  cc.  is  de- 
sired draw  water  up  to  the  top  mark  and  let  out  into  the  plate  or 
tube  until  the  lowest  graduation  marked  1  cc.  is  reached.  This 
gives  1  cc.  of  water.  When  fractional  parts  of  a  cubic  centimeter 
are  desired  draw  water  to  the  top  mark  and  let  out  till  it  reaches 
the  graduation  marked  with  the  number  of  tenths  required.  Each 
of  the  divisions  represents  .1  cc.  One  pipette  is  required  for  each 
sample.  In  handling  pipettes  never  let  the  lower  part  touch  any- 
thing but  the  sample  to  be  tested.  Keep  the  fingers  off  from  it. 
Let  the  case  of  pipettes  lie  on  the  table — never  stand  it  on  end. 

49 


Laboratory  Outline  for  General  Bacteriology 


Pour  into  the  plates  melted  agar  cooled  to  40°  C.  and  mix  well 
with  the  water. 

When  hard  place  plates  and  fermentation  tubes  in  37°  C. 
incubator. 

Incubate  two  days.  They  are  then  ready  to  be  studied.  Meas- 
ure the  percentage  of  gas  by  the  gasometer  chart  on  page  41. 

Count  the  numbers  of  bacteria  on  the  plates  using  the  ruled 
glass  counting  plate  and  a  hand  lens.  Make  an  average  of  the  four 
plates  before  stating  the  number  of  bacteria  per  cubic  centimeter. 
To  make  this  average  multiply  the  .1  cc.  plate  count  by  10,  the 
.2  cc.  by  5,  and  the  .5  cc.  by  2  before  adding  to  the  1  cc.  plate  count. 
Divide  by  four. 

Gas  percentages  are  not  averaged.  Results  are  stated  in  num- 
ber of  tubes  showing  more  than  10  (/(  of  gas. 


BACTERIA  IN  MILK 

Samples  are  taken  from  well  mixed  milk  into  sterile  bottles 
and  tested  as  soon  as  possible.  Samples  that  cannot  be  tested  at 
once  should  be  kept  cold — below  40°  F.  Standard  agar  is  used 
for  plating. 

Milk  cannot  be  added  directly  to  the  plates  as  is  done  in  plat- 
ing ordinary  water  samples  because  it  usually  contains  so  many 
bacteria  that  the  colonies  would  not  have  room  to  develop  properly 
or  be  readily  counted.  Also  the  milk  would  cloud  the  agar  so  the 
colonies  could  not  be  easily  seen.  By  diluting  the  milk  with  ster- 
ile water  clear  plates  having  between  25  and  300  colonies  can 
usually  be  obtained.  Plates  having  numbers  of  colonies  outside 
these  limits  do  not  give  as  reliable  results.  Four  plates  are  made 
using  at  least  two  different  dilutions.  For  miscellaneous  milk 
samples  the  character  of  which  is  not  known  dilutions  may  be 
made  ranging  from  1-100  to  1-10000.  When  something  is  known 
as  to  the  quality  of  the  milk,  dilutions  are  made  so  that  the  highest 
one  will  give  about  100  colonies  to  the  plate.  For  a  sample  of  milk 
which  may  contain  20,000  bacteria  per  cc.  dilutions  of  200  and  100 
would  be  best  for  giving  the  desired  numbers  of  colonies  on  the 
plates.  The  method  of  plating  is  as  follows:  Shake  the  milk 
sample  vigorously  25  times.  With  a  sterile  pipette  transfer  1  cc. 
to  dilution  bottle  containing  199  cc.  of  sterile  water.  As  this 
pipette  is  to  be  used  once  later  it  is  laid  aside  so  that  the  lower 
half  will  touch  nothing.  Shake  dilution  25  times  and  with  another 
sterile  pipette  transfer  1  cc.  to  each  of  two  plates.  This  gives  the 
200  dilution.  Lay  aside  this  pipette  also  so  it  will  not  become  con- 

50 


Laboratory  Outline  for  General  Bacteriology 

laminated.  With  the  first  pipette  used  transfer  another  cubic 
centimeter  of  milk  to  the  dilution  bottle.  This  doubles  the  amount 
of  milk  and  decreases  the  dilution  one-half,  thus  giving  the  100 
dilution.  Discard  the  pipette.  Shake  sample  and  make  transfers 
from  it  to  the  remaining  two  plates,  using  the  same  pipette  as  was 
used  for  the  first  plates  but  first  rinsing  out  any  of  the  first  dilu- 
tion remaining  in  it  by  drawing  up  some  of  the  second  dilution 
and  letting  it  out.  Transfers  are  always  made  using  1  cc.  quanti- 
ties in  dilution  work.  Melted  agar  is  poured  into  the  plates  and 
mixed  with  the  dilution.  Plates  are  inverted  after  hardening  and 
incubated  at  37°  C.  for  two  days. 

Now  if  the  sample  of  milk  is  older  or  of  poorer  quality  and 
may  contain  500,000  bacteria,  allowing  100  bacteria  per  plate 
would  make  the  required  dilution  5000.  This  is  too  large  a  quan- 
tity of  water  to  be  handled  in  one  container.  The  same  result  is 
secured  by  using  two  bottles  in  succession  having  50  cc.  and  100  cc. 
of  water  respectively.  Multiplying  amounts  of  water  in  the  bottles 
gives  the  dilution.  When  bottles  of  different  sizes  are  used  always 
use  the  smaller  first.  In  this  case  1  cc.  is  taken  from  milk  sample 
to  50  cc.  bottle  and  pipette  discarded.  This  50  cc.  dilution  is  used 
exactly  the  same  as  the  milk  sample  was  in  the  preceding  case. 
Laboratory  dilution  bottles  may  be  had  marked  for  10  cc.,  20  cc., 
40  cc.,  50.,  60.,  100.,  200.,  400,  and  500  cc. 

After  incubation  counts  are  made  from  the  plate  cultures.  To 
find  the  number  of  bacteria  per  cc.  of  milk,  multiply  the  plate 
counts  by  their  respective  dilutions,  add  together  and  divide  by 
four.  If  some  plates  are  covered  entirely  by  spreaders  or  other- 
wise spoiled  they  are  left  out  of  the  average. 

When  it  is  desired  to  find  the  number  of  acid  producing  bac- 
teria and  liquefying  bacteria  in  milk,  litmus  lactose  gelatin  plates 
are  made  in  the  same  manner  as  the  agar  plates  except  that  2  cc. 
of  litmus  solution  is  added  to  the  gelatin  just  before  pouring  plates. 
The  plates  are  incubated  right  side  up  at  20°  C.  for  seven  days 
unless  liquefying  bacteria  make  it  necessary  to  count  earlier.  Acid 
bacteria  turn  blue  litmus  red. 

The  interpretation  of  the  results  must  depend  upon  the  his- 
tory of  the  milk. 

1.  When  analysis  is  made  immediately  after  milking  con- 
clusions may  be  drawn  as  to  the  cleanliness  and  care  in  the  dairy, 
the  thoroness  in  cleaning  and  sterilizing  of  the  utensils,  and  some- 
times as  to  the  presence  of  cows  with  infected  udders.  With  prop- 
erly cleaned  and  sterilized  milk  vessels  and  care  in  milking  the 
numbers  of  bacteria  should  not  exceed  10,000,  and  may  easily  be 
brought  lower.  Larger  numbers  indicate  unclean  methods,  un- 
sterile  utensils  or  infected  udders. 

51 


Laboratory  Outline  for  General  Bacteriology 


2.  If  milk  is  properly  cooled  with  ice  the  numbers  should 
increase  only  very  slowly.     50,000  is  a  high  count  for  milk  deliv- 
ered the  next  day  even  in  warm  weather. 

3.  For  milk  which  must  be  a  longer  time  in  transportation 
higher  numbers  may  be  expected.    Milk  with  more  than  1,000,000 
bacteria  has  not  been  properly  cared  for. 


DIRECT  MICROSCOPIC  EXAMINATION  OF  MILK 

The  advantages  of  this  method  are  that  it  is  very  rapid,  re- 
sults being  obtained  within  a  few  minutes  and  before  the  milk 
from  which  the  sample  is  taken  is  used.  It  is  simple  and  requires 
little  apparatus.  It  also  gives  some  idea  as  to  the  kinds  of  bac- 
teria. It  does  not,  however,  distinguish  between  the  living  and 
the  dead  bacteria.  With  milk  of  fairly  good  quality  the  numbers 
cannot  be  easily  determined  as  no  bacteria  can  be  found.  Its 
chief  advantage  is  in  the  quick  detection  of  milk  of  very  poor 
quality. 

The  milk  samples  are  taken  and  shaken  as  for  plating.  With 
pipette  calibrated  to  deliver  1-100  cc.,  transfer  this  amount  of 
milk  to  a  glass  slide.  Spread  over  just  1  sq.  cm.  The  slide  may 
have  the  square  centimeter  ruled  on  it  or  it  may  be  placed  over 
glass  or  paper  ruled  in  centimeter  squares  to  serve  as  a  guide. 
Duplicate  smears  should  be  made  on  the  same  slide.  Milk  on  the 
exterior  of  the  pipette  should  be  wiped  off  before  making  the 
smear.  Pipettes  do  not  need  to  be  sterilized.  They  are  rinsed 
out  with  water  between  samples.  The  water  remaining  is  rinsed 
out  in  the  next  milk  sample.  Dry  the  smears  without  heat.  Cover 
smears  with  xylol  for  1  minute.  Drain  and  dry.  Cover  with  95% 
alcohol  for  1  minute  or  more  to  fix  the  film  to  the  slide.  Drain  and 
stain  with  a  fresh  aqueous  solution  of  methylene  blue  for  10 
minutes.  Wash.  If  film  is  too  dark  decolorize  slightly  with  alco- 
hol. Slides  may  be  examined  at  once  or  kept  for  later  reference. 
They  are  examined  under  the  microscope  using  the  oil  immersion 
objective.  The  bacteria  in  at  least  30  fields  in  a  square  centimeter 
should  be  counted.  With  the  Leitz  microscope  there  are  about 
4800  fields  in  a  square  centimeter ;  with  the  Bausch  &  Lomb  5,400. 
Since  only  1-100  cc.  of  milk  is  used  the  number  of  bacteria  is  found 
by  multiplying  the  average  number  of  bacteria  per  field  by  the 
number  of  fields  in  1  sq.  cm.  and  this  by  100. 


Laboratory  Outline  for  General  Bacteriology 

EFFECT  OF  TEMPERATURE  ON  KEEPING  QUALITY  OF  MILK 

Absolutely  fresh  milk  of  good  quality  is  taken  at  5  P.  M.  and 
after  mixing  well  is  divided  into  three  parts.  These  are  kept  at 
5°  C.,  20°  C.,  and  37°  C.  respectively  for  twenty  hours. 

Make  four  litmus  lactose  gelatin  and  four  agar  plates  from 
each  sample  using  the  following  dilutions  for  each  medium : 

5°  C 

200     200     100     100 

20°  C. 
100,000     100,000     50,000     50,000 

37°  C 

1,000,000     1,000,000     500,000     500,000 

Incubate  agar  plates  at  37°  C.  for  two  days.  Count  and  find 
the  average  number  of  bacteria  per  cubic  centimeter  for  the  three 
samples. 

Incubate  the  litmus  lactose  gelatin  plates  at  20°  C.  for  seven 
days  unless  liquefiers  threaten  to  destroy  the  plates  when  they 
should  be  counted  sooner.  Count  total  number  of  bacteria,  acid 
bacteria  (those  that  turn  litmus  around  them  pink)  and  liquefying 
bacteria.  Calculate  the  average  number  per  cubic  centimeter  for 
total  bacteria,  acid  bacteria,  and  liquefying  bacteria. 

How  do  the  total  numbers  on  agar  and  gelatin  compare? 


BACTERIA  FROM  VARIOUS  SOURCES 

The  purpose  of  these  experiments  is  to  show  the  abundance 
of  bacteria  in  their  relation  to  our  everyday  life. 

1.  Wash  hands  in  pan  containing  1000  cc.  of  sterile  water. 
Plate  using 

1  cc.  .5  cc.  .2  cc.  .1  cc. 

2.  Wash  hands  carefully  with  soap  under  tap  then  without 
drying  them  or  touching  anything  wash  again  in  a  second  pan  con- 
taining 1000  cc.  of  sterile  water.     Plate  using 

1  cc.  .5  cc  .2  cc.  .1  cc. 

3.  An  apparently  clean  wash  bowl  is  washed  with  a  sterile 
brush  and  1000  cc.  of  sterile  water.     Plate  using 

1  cc.  .5  cc.  .2  cc.  .1  cc. 

4.  Place  a  pencil  in   test  tube  with  10  cc.  of    sterile  water. 
Wash  pencil  well  and  plate  using 

1  cc.  .5  cc.  .2  cc.  .1  cc. 

53 


Laboratory  Outline  for  General  Bacteriology 

5.  Pour  three  plates  with  agar.    When  cool  sneeze  into  one, 
cough  into  another  and  place  a  hair  in  the  third. 

6.  Catch  two  flies  and  place  them  in  bottle  with  100  cc.  of 
sterile  water.    Shake  5  minutes  and  plate  using 

1  cc.  .5  cc.  .2  cc.  .1  cc. 

7.  Shake  a  dishcloth,  which  has  been  used  twice  in  ordinary 
way  since  being  thoroughly  boiled,  in  1000  cc.  of  sterile  water. 
Plate  using 

1  cc.  .5  cc.  .2  cc.  .1  cc. 
Pour  agar  in  plates  and  incubate  2  days  at  37°  C.  and  study. 

8.  Flame  a  clean  glass  slide,  cool,  and  touch  lip  to  it  as  much 
as  you  would  touch  it  to  a  glass  in  drinking,    Dry,  and  pass  three 
times  through  flame.    Stain  1  minute  with  watery  fuchsin.    Wash, 
dry  and  examine  under  the  microscope  using  the  oil  immersion 
objective.     Note  the  forms  and  arrangement  of  the  bacteria  pres- 
ent and  the  approximate  number  of  bacteria  per  field. 

9.  Sterilize  platinum  loop,  cool,  scrape  along  the  teeth  near 
the  gums.    Mix  with  drop  of  water  on  a  slide.     Spread,  dry,  flame 
three  times,  stain  1  minute  with  watery  fuchsin,  wash,  dry,  and 
examine  under  the  miscroscope  using  the  oil  immersion  objective. 
Note  the  forms,  arrangement,  and  abundance  of  the  bacteria. 


DISTILLED  WATER  EXPERIMENT 

To  demonstrate  the  small  amount  of  food  needed  by  bacteria, 
and  that  distilled  water  is  not  the  same  as  sterile  water. 

Draw  off  100  cc.  of  distilled  water  into  each  of  two  sterile 
flasks.  Mark  them  "A"  and  "B". 

Sterilize  flask  "A"  in  autoclav  for  15  minutes  at  10  pounds 
pressure.  When  cool  make  an  agar  plate  from  each  flask  using 
1  cc.  for  inoculation. 

Inoculate  flask  A  with  a  very  small  amount  of  B  coli.  Shake 
and  plate  1  cc.  in  agar. 

Keep  flasks  and  plates  in  locker. 

After  five  days  count  plates  and  make  others  from  flasks 
using  dilutions  of  1-10,  1-100,  and  1-1000. 

After  5  days  count  and  find  whether  there  has  been  an  in- 
crease or  decrease  in  the  numbers  of  bacteria. 


Laboratory  Outline  for  General  Bacteriology 


BACTERIA  IN  MEATS 

Meats  are  brought  directly  from  the  market  to  the  laboratory. 
Those  not  already  ground  are  passed  through  sterilized  meat 
grinders.  Weigh  out  5  grams  on  sterile  paper.  Place  in  warm 
mortar.  Add  enough  sterilized  quartz  sand  to  grind  well.  After 
grinding  well  add  little  by  little  95  cc.  of  warm  (about  38°  C.) 
sterilized  water.  This  makes  a  dilution  of  20.  Use  bottles  for  the 
further  dilutions  required.  For  Hamburg  steak  and  sausage  use 
dilutions  of  200,000  and  100,000;  for  mutton,  veal,  and  pork 
use  100,000  and  50,000.  Make  four  agar  plates  from  each  sample. 
Incubate  at  37°  C.  for  two  days.  Count  plates  and  find  the  number 
of  bacteria  per  gram  of  meat. 


TESTING  FOR  PRESENCE  OF  SPORES 

Inoculate  tubes  of  sterile  nutrient  broth  with  the  following : 

1.  Potato  parings 

2.  Hay 

3.  Corn  meal 

4.  Hamburg  steak 

5.  Oatmeal 

6.  Wheat  meal 

7.  Beans 

8.  Old  milk 

9.  Mornings  milk 

10.  Sweet  corn 

11.  Raisins 

After  inoculating  place  in  steam  sterilizer  in  flowing  steam 
for  15  minutes.  This  kills  all  vegetative  forms.  Incubate.  After 
seven  days  examine  tubes  and  make  records  of  presence  or  ab- 
sence of  growth. 


CONVERSION  FACTORS 

Grams  to  grains  multiply  by  15.432 

Grams  to  ounces  avoirdupois 
Kilograms  to  pounds 


Cubic  centimeters  to  fluid  ounces  imperial 
Liters  into  fluid  ounces  imperial 
Meters  into  inches 

55 


0.03527 

2.2046 

0.0352 

35.2 

39.37 


Laboratory  Outline  for  General  Bacteriology 

Grains  into  grams 

Avoirdupois  ounces  into  grams 

Troy  ounces  into  grams 

Fluid  ounces  into  cubic  centimeters 

Pints  into  liters 

Inches  into  meters 

Degrees  C.  X  1.8  4-  32  =  degrees  F. 
Degrees  F.  —  32  —  1.8  ==  degrees  C. 


0.0648 
28.35 
31.104 
28.42 
0.568 
0.0254 


COMPARISON  OF  METRIC  AND  ENGLISH  SYSTEMS 

Meter=39.3704  inches. 

Millimeter  =  0.03937  inches   (0.04  approximately) 

Inch  ==25.3997  mm.  (25.4  approximately) 

Liter  =  2. 11  pints  (1  quart  approximately) 

Cubic  centimeters  =  16.23  minims. 

Fluid  ounce^=29.578  cubic  centimeters  (30  cc.  approximately) 

Gram  =  15.432  grains 

Kilogram  =  2.204  avoirdupois  pounds 

Ounce  avoirdupois  —  28.349  grains 

Pound  avoirdupois  =  453.584  grams 

Ounce  troy  =  31.103  grams 

Square  centimeter  —  0.1548  square  inches 

Square  inch  —  6  square  centimeters  (approximately) 

Cubic  centimeter  =  0.0609  cubic  inches 

Cubic  inch  =  16  cubic  centimeters  (approximately) 


56 


Appendix 


Glossary  of  Laboratory  Bacteriological  Terms 


Acid  curd, 
Adherent, 

Aerobic 

Amorphous 
Anaerobic, 

Anaerobic, 
Anaerobic, 

Arborescent, 
Autotrophic 


Beaded, 

Bipolar, 
Brittle, 
Butyrous, 
Capsule, 

Chains, 

Chromogenesis, 
Chromogenic, 
Chromogen, 
Ciliate, 

Clavate, 
Coagulate, 

Commas, 
Concave, 
Concentric, 


Contoured, 

Convex, 
Coriaceous, 

Crateriform, 
Cretaceous, 
Cuneate, 
Dextrose, 


a  curd  produced  by  an  acid. 

applied  to  the  sporangium  wall,  when  it  continues 
to  enclose  the  developed  spore. 
(Strictly),  growing  only  in   the   presence   of  free 
oxygen. 

without  visible   differentiation  in  structure, 
growing  in  the  absence  of  free  oxygen ;  cannot  grow 
in  presence  of  oxygen. 

(facultative),  growing  both  in  the  presence  and 
absence  of  free  oxygen. 

(strictly),  growing  only  in  the  absence  of  free 
oxygen. 

branched,  tree-like  growth. 

obtaining  their  atomic  elements  carbon,  oxygen, 
hydrogen,  nitrogen,  sulphur,  and  iron  from  inor- 
ganic compounds  or  molecules. 

in   stab  or  stroke   culture,  disjointed   or  semi-con- 
fluent colonies  along  the  line  of  inoculation, 
at  both  poles  or  ends  of  the  bacterial  cell, 
growth  dry,  friable  under  the  platinum  needle, 
growth  of  butter-like  consistency, 
a   gelatinous    envelope    surrounding   bacteria    (not 
easily  stained.) 

short  chains  contain  from  2  to  8  elements,  Long 
chains  more  than  8  elements, 
the  production  of  color, 
relating  to  or  having  chromogens. 
any  organic  coloring  matter. 

having  fine,  hair  like  extensions,  resembling  cilia, 
sometimes  not  visible  to  the  naked  eye. 
club-shaped.  (Usually  an  involution  form.) 
to  curdle  or  clot  by  chemical  action  or  fermenta- 
tion. 

shaped  like  a  comma, 
curving  in. 

having  a  common  center,  as  spheres  or  circles;  said 
loosely  of  any  curves  that  are  parallel  or  nearly 
so. 

an  irregular,  smoothly  undulating  surface,  like  that 
of  a  relief  map. 
curving  out. 

growth  tough,  leathery,  not  yielding  to  the  plati- 
num needle. 

the  crater-shaped  liquefaction  of  a  medium, 
growth  opaque,  and  white,  chalky, 
wedge-shaped, 
grape  sugar,  glucose. 

57 


Laboratory  Outline  for  General  Bacteriology 


Diastatic  action, 

Drum  sticks, 
Echinulate, 

Effuse, 

Elongated, 

Endospore, 


Entire, 

Filamentous, 

Filaments,- 

Filiform, 

Flagella, 

Flaky, 

Flocculent, 

Fluorescent, 

Granular, 
Gram's  stain, 


Grumose,  . 
Heterotrophic, 


Infundibuliform, 
Iridescent, 

Lactose, 
Litmus, 
Liquefaction, 
Lobate, 

Long  rod, 
Luminous, 
Maximum  temperature, 

Membranous, 
Minimum  temperature 

Motile, 
Myceloid, 

Napiform, 

Non-chromogenic, 

Opaque, 


conversion    of   starch    into     simple     carbohydrates, 
such  as  dextrins  and  sugar  by  means  of  diastase, 
bacteria  in  the  shape  of  a  club. 

a  growth  along  the  line  of  inoculation  with  toothed 
or  pointed  margins. 

growth  thin,  veilly,  unusually  spreading, 
drawn  out,  lengthened  or  extended, 
thick  walled  spores  formed  within  the  bacterial 
cell;  i.  e.  typical  bacterial  spores  like  those  in  B. 
anthracis  or  B.  subtilis. 

smooth,  having  a  margin  destitute  of  teeth  or 
notches. 

growth  composed  of  long,  irregularly  placed  or 
interwoven  threads. 

applied  to  the  morphology  of  bacteria,  it  refers  to 
thread-like  forms,  generally  unsegmented;  if  un- 
segmented,  to  be  distinguished  from  chains  (q.  v.) 
by  the  'absence  of  constrictions  between  the  seg- 
ments. 

in  stroke  or  stab  cultures,  a  uniform  growth  along 
the  line  of  inoculation. 

fine  hair-like  extensions  that  propel  certain  kinds 
of  bacteria  through  liquids. 

resembling  or  consisting  of  flakes  or  separable  into 
flakes. 

containing   small   adherent    masses    of   bacteria    of 
various  shapes  floating  in  the  culture  fluid, 
having  one  color  by  transmitted  light  and  another 
by  reflected  light, 
composed  of  small  granules. 

a   method    of    differential    bleaching    after    gentian 
violet,  methyl  violet,   etc.      The   +   mark   is  to   be 
given  only  when  the  bacteria  are  deep  blue  or  re- 
main blue  after  counterstaining. 
clotted. 

obtaining  their  elemental  atoms  of  carbon,  oxygen, 
hydrogen,  nitrogen,  sulphur  and  iron  from  organic 
compounds. 

form  of  a  funnel  or  inverted  cone, 
exhibiting    changing    rainbow    colors    in    reflected 
light. 

milk  sugar. 

red  indicates  acid,  blue  indicates  alkaline, 
change  of  a  solid  into  a  liquid. 

having  the  margin  deeply  undulate,  producing  lobes, 
(see  undulate.) 

more  than  two  diameters  in  length, 
glowing  in  the  dark,  phosphorescent, 
the  temperature  above  which  growth  does  not  take 
place. 

growth  thin,  like  a  membrane. 

temperature  below  which  growth  does  not  take 
place. 

having  power  of  motion. 

colonies   having   the   radiately   filamentous   appear- 
ance of  mold  colonies, 
liquefaction  in  the  form  of  a  turnip, 
not  having  chromogens  or  coloring  matter, 
impervious  to  light. 

58 


Laboratory  Outline  for  General  Bacteriology 


Opalescent, 

Optimum  temperature. 

Papillate. 

Pathogen, 

Pellicle, 

Peritrichiate, 
Peptonization, 

Persistent, 

Photogenic, 

Plumose, 

Polar, 

Prototrophic, 


Pulvinate, 
Punctiform, 

Pure  culture 
Rapid, 
Raised, 
Reaction, 


Reduction, 


Rennet  curd, 
Rhizoid, 

Ring, 

Rods, 
Rugose, 
Saccharose 
Saccate, 

Saprophyte, 
Short  rods, 
Slow, 
Spirals, 
Spreading, 

Sporangia, 
Sporangium  wall, 

Spore, 
Stratiform, 

Sucrose, 

Transient, 

Translucent, 

Truncate, 

Turbid, 

Umbonate, 

Tndulate, 

Villous, 

Viscid 


resembling  the  color  of  an  opal, 
the  temperature  at  which  growth  is  most  rapid, 
growth  beset  with  small  nipple  like  processes, 
a  kind  of  bacteria  causing  disease, 
bacterial   growth   forming   either   a   continuous    or 
interrupted  sheet  over  the  culture  fluid, 
covered  with  flagella  over  the  entire  surface, 
rendering  albumen  of  milk  soluble  by  the  action  of 
trypsin. 

lasting  many  weeks  or  months, 
producing  phosphorescence, 
a  fleecy  or  feathery  growth, 
at  the  end  of  pole  of  the  bacterial  cell, 
prototrophic  bacteria  are  those  that  can  use  elemen- 
tal atoms  of    carbon,  oxygen,    hydrogen,    nitrogen, 
sulphur  and  iron. 

decidedly  convex,  in  the  form  of  a  cushion, 
very  small,  but  visible  to  the  naked  eye;  under  one 
mm.  in  diameter. 

culture  containing  only,  one  species  of  bacteria, 
developing  in  24  to  48  hours, 
growth  thick,  with  abrupt  or  terraced  edges, 
refers  to  the  production  of  acid  or  alkali  in  culture 
media  by  bacteria.   Use  -+-  to  indicate  acid,  —  to  in- 
dicate alkaline,  and  O  no  change, 
removing  oxygen  from  a  chemical  compound. 
Refers  to  the  conversion  of  nitrate  to  nitrite,  am- 
monia, or  free  nitrogen,  and  to  the  decolorization 
of  litmus. 

a  curd  produced  by  a  lab  ferment,  not  by  an  acid, 
growth  of  an  irregular  branched  or  root-like  char- 
acter, as  in  B.  mycoides. 

growth   at  the   upper   margin    of   a   liquid    culture, 
adhering  more  or  less  closely  to  the  glass, 
long,  more  than  two  diameters  in  length, 
wrinkled, 
cane  sugar. 

liquefaction  in  the  form  of  an  elongated  sac,  tubu- 
lar cylindrical. 

an  organism  living  on  dead  organic  matter, 
less  than  two  diameters  in  length, 
requiring  5  or  6  days  for  development, 
bacteria  growing  in  the  form  of  a  spiral, 
growth  extending  much  beyond  the  line  of  inocu- 
lation, i.   e.  several  millimeters  or  more, 
cells  containing  endospores. 

the  wall  of  a  cell  within  which  a  spore  has  been 
produced. 

the  resting  stage  of  bacteria. 

liquifying  to  the  walls  of  the  tube  at  the  top  and 
then  proceeding  downwards  horizontally, 
same  as  saccharose, 
lasting  a  few  days, 
semi-transparent, 
ends  abrupt,  square. 

cloudy  with  flocculent  particles;  i.  e.  cloudy  plus 
flocculence. 

having  a  button-like,  raised  center, 
border  wavy,  with  shallow  sinuses, 
having  hair-like  extensions. 

growth  follows  the  needle  when  touched  and  with- 
drawn; sediment  on  shaking  rises  as  a  coherent 
swirl. 

59 


Index 


Acid,  production  of . . .  .31,  40,  51,  53 

Adjustment,    coarse 11 

fine   11 

Aerobic  bacteria,  strict    31,  32,  39,  57 

Agar 

composition    of 17 

melting  point  of 17,  18 

nitrate 31,  34,  39 

slant 20,  29,  32,  34,  37 

solidification  of 18 

source    of 17 

starch 31,  34,40 

types  of  growth  on.  .  .29,  30,  32,  33 
use  of  17,  18,  22,  23,  38,  49,  51,  54, 
55 

Air,  bacterial  analysis  of 18-20 

Alcohol,  cleaning  of  microscope.  . .  .11 

for    decolorization 35,   52 

for  stains 35,  52 

Anaerobic,  facultative 32,  39,  57 

strict... 31,  32,  39,  57 

Anilin  gentian  violet, 

preparation  of  35 

use    of 35 

Apparatus,  sterilization  of....  13,  14 

Area  of  Petri  dish, 

determination  of 19 

Autoclav    14 

Azotobacter    chroococcum 46 

B 

Bacteria  (B.  and  Bact.  listed 
together) 

abortus    46 

acidophilus    45 

aerogenes 45,    46 

albolactus    44 

arborescens 44 

amylovorus   45 

aterrimus    44 

aquatilis    45 

buccalis    44 

butyricus     44 

campestris     45 

cereus     44 

cohaerens    45 

coli    45 

coscoroba     46 

cyaneus    44 

cyanogenes    45 

enteritidis    45 


Bacteria  (continued) 

erythrogenes 45 

fluorescens 45 

fluorescens  liquef  aciens    45 

fluorescens  non  liquef  aciens 45 

fluorescens  tenuis 45 

f usif ormis   44 

gasoformans 45 

graveolens 44 

Grunthal  45 

Hartlebii  45 

icteroides     45 

lactis    44 

lactis  acidi   43 

lactis  erythrogenes   45 

megatherium    44 

mesentericus     44 

mirabilis    45 

moelleri    44 

mucosus 45 

mycoides    44 

niger    44 

niger  lactis 44 

petasites    44 

prodigiosus   45 

proteus     45 

proteus  vulgaris .45 

proteus  Zopfii    44 

pullorum    46 

radicicola     45 

simplex    , 44 

subtilis 44 

subtilis  ruber   44 

terminalis    44 

tumef  aciens    45 

vulgatus    44 

Bacteria,  effect  of  moist 

and  dry  heat   13,  14 

flagella  of   17 

from  various  sources   53 

Gram   negative    36,   58 

Gram  uositive 36,  58 

identification  of  22,  23,  24,  25,  26, 

27.  28,  29,  30.  31.  32 

in  air  18,  19,  20 

in  meats    55 

on  pencils   53 

on  hands   53 

spores  of 14,  55 

unstained,  examination  of  .  .  .16,  17 
vegetative  forms,  destruction 

of,  by  heat 14 

Broth,  nitrate 38,  39 

nutrient  38 


60 


Laboratory  Outline  for  General  Bacteriology 


Calculations 

for  microscopic  counts 52 

for  plate  counts 19 

Capsules    57 

Carbol  f uchsin 35 

Cautions  for  bacteriological  work  .  .  . 

22,  23,24,  25,26,  27 

Chart,  descriptive  (Soc.  Am.  Bact)  .  . 

28,  29,  30,  31,  32 

Coagulation    40,  57 

Condensation  water    17 

Condenser,  microscope  11 

Conversion,  Metric  to  English  ...  .55 

English  to  Metric 56 

Cough,  bacteria  disseminated  by  . .  .54 
Counting, 

direct  microscopic   52 

lens     19 

plates 19 

Cover-glass 16,  36 

Culture  media,  descriptions  of  ...  17, 

18,37,38,39,40 

Cultures, 

age  for  examination 34 

plate    24,  25 

pure    59 

slant 33 

stab    3£ 

streak     33 

Curd,  acid 31,57 

rennet   31,  59 

D 

Death-point,  thermal 41 

Descriptions  of  bacteria   ..42,  43,  44, 
45,  46 

Descriptive  chart 43,  44,  45,  46 

Dextrose  fermentation  tubes 34 

Diastatic    action    58 

Dilution  methods  of 50,  51 

Dishcloth,  bacteria  on    54 

Dish,  Petri 15,  38,  49 

Distilled  water  experiment   54 

Do's  in  identification  of  unknown 

cultures    22,  24,  26 

Don'ts    in    identification    of   unknown 

cultures 23,  25,  27 


E 


Ehrlich's  test  for  indol 39 

Endpspores   29,  37,  58 

Equipment  of  lockers    14,  15 

Examination  of  cultures,  age  for  .  .34 


Facultative  anaerobic 32,  39,  57 

Fermentation  tubes   39 

chart  for  measuring  gas 41 

Films,  preparation  of 36 

Flies,  bacteria  on 54 

Flagella,  method  of  staining 37 

Flame,  gas  for  sterilization   13 

Focusing 11 

Form,  for  writing  up  exercises  ...  .15 
Formulae,  for  changing  °C.  to  °F..  .56 
Metric  to  English,  vice  versa.  .55,  56 
Fuchsin 

alcoholic  solution  of 35 

carbol    35 

watery 35 


Gelatin,  history  of 17 

constituents  of   17 

litmus  lactose 51,  53 

liquefaction  of 32,  33 

plating   24,  25 

solidifying  points  17 

stabs,  types  of  growth 33 

Gentian  violet 

alcoholic  solution  of 35 

anilin   water    35 

watery 35 

Glassware,  sterilization  of 14 

Glossary, 57,  58,  59 

Glycerine  fermentation  tubes 31 

Gram  negative 36,  58 

positive 36,  58 

Gram's  stain   35 

mixture  35 


II 


Hair,  bacteria  on   .54 

Hands,  bacteria  on    53 

Heat,  dry  for  sterilizing 14 

moist,  for  sterilizing    14 

Hot  air  sterilization 14 

Hydrogen  sulphide,  test  for 34,  39 


Illumination  for  microscope 11 

Incubation,  temperatures  of 34 

Indicator 40 

Indol    39 

Ehrlich's  test  for 39 

Inoculation   of  media    

22,  23,  24,  25,  26,  27 


61 


Laboratory  Outline  for  General  Bacteriology 


Lactose  fermentation  tubes 31 

Lead  acetate  agar 34,  39 

Light,  effect  on  bacteria  25,  38 

for  microscopic  work  11 

Lips,  bacteria  on  54 

Liquefaction  of  gelatin  32,  33 

types  of  33 

Litmus  milk  40 

reduction  of 40 

Lockers,  equipment  of 14,  15 

Loeffler's  methylene  blue 35 

M 

Magnification 12 

Meats,  bacteria  in 55 

Media,  liquid    24,  25 

nutrient    38 

solid 37 

Method  for  determining  the  per 
cent  of  bacteria  killed  by  sun- 
light   38 

Methylene  blue   35 

Micrometer  scale   11 

Measurement  of  bacteria 12 

Micrococcus  agilis  12,  43 

Micrococcus  lactis  varians 43 

Microscope   11 

Abbe  condenser 11 

cleaning  of 11 

how  to  use 11 

lighting   for    11 

magnification    12 

mirror   of    11 

Milk,  dilutions  for  plating   ...  .50,  51 
direct  microscopic  examination  .  .  52 

effect  of  temperature  on    53 

peptonization  of 32,  40 

reaction  of 40 

staining  of 52 

Mirror,  concave 11 

plane    11 

Molds    19 

Mordant  for  flagella  staining 3~ 

Morphology    36 

Motility   16,  17 

Mouths  of  culture  tubes,  flasks, 

sterilization  of 22,  23 

N 

Nitrate  agar 38 

Nitrate  broth    38 

test   solution  for    39 

Nitrites,  test  solution  for   .39 

Nutrient  broth    .  .38 


O 

Objectives,  oil  immersion  11 

cleaning  of 12 

Oil,  cedar  11 

immersion  .  .11 


Paradimethylaminobenzaldehyde    .  .39 

Pencils,  bacteria  on  53 

Peptone     39 

Peptonization  of  milk   40 

Pipette   15,  49 

Plates,  agar 38 

gelatin     38 

Plate  cultures,  examination  of  .  .50,  51 

Plating 24,25,  26,  27 

Platinum  needle,  sterilization  of...  14 
Potato 

preparation  of 37 

types  of  growth  on 33 

Pressure,  of  steam  in  sterilization  .  .14 
Prototrophic     59 

R 

Reaction   59 

Reduction  of  litmus 40 

Rennet  curd 31,  59 

Resistance  of  spores 55 

Results,  interpretation  of    51 

Rhodoccus  roseus 43 

Rules  for  use  of  microscope   11 

S 

Saccharose  fermentation  tubes  ...  .31 

Sarcina 

aurantiaca    43 

flava     43 

lutea    43 

Slanecii   43 

Sneeze,  bacteria  disseminated  by  .  .  54 

Spores,  testing  for  presence  of  ...  .55 

Staining 

flagella    37 

Gram's  method   35 

Stains,  preparation  of 3^ 

Staphlococcus  albus 43 

Starch,  agar 

test  for  diastatic  action  of  bac- 
teria  on    40 

Steam,   sterilization   by    14 

Sterilization 

by  dry  heat    14 

by  gas  flame  14 

by  steam  under  pressure   14 


62 


Laboratory  Outline  for  General  Bacteriology 


Streptococcus  hemolyticus 43 

lactis  43 

rheunnaticus  43 

Sunlight,  effect  on  bacteria 38 


Teeth,  bacteria  on 54 

Temperature,    conversion    of    de- 
grees of,  effect  on  bacteria 53 

Thermal   death   point    41 

Testing    for    spores     in    various 

foods   55 

Time  for  sterilization  14 

requirements  of  cultures 34 

requirements  of  stains 35,  36 

Tubes,  fermentation   31,  39 


W 

Washbowl,  bacteria  on 53 

Water,  bacterial  analysis  of 49 

distilled    54 

Whey,  extrusion  of 40 


Xylol,  use  of 11 


Yeasts,  examination  of 17 


Zone,  clear,  produced  by  diastatic 
enzyme 40 


63 


UNIVERSITY  OF  CALIFORNIA  LIBRARY 


